Antibodies Directed to Angiopoietin-1 and Angiopoietin-2 and Uses Thereof

ABSTRACT

Disclosed are specific binding agents, such as fully human antibodies, that bind to angiopoietin 1 and/or angiopoietin-2. Also disclosed are heavy chain fragments, light chain fragments, and CDRs of the antibodies, as well as methods of making and using the antibodies.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/194,854 filedJul. 29, 2011, now allowed, which is a divisional of U.S. Ser. No.12/378,993 filed Feb. 9, 2009 and now U.S. Pat. No. 8,030,025 issued onOct. 4, 2011, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/139,361 filed Dec. 19, 2008, and U.S. ProvisionalApplication Ser. No. 61/061,943 filed Jun. 16, 2008, and U.S.Provisional Application Ser. No. 61/066,632 filed Feb. 20, 2008, whichare incorporated herein by reference.

The present application is being filed along with a Sequence Listing intext format. The Sequence Listing is provided as a file entitledA-1382-US-CNT_SeqListingAsFiledInParent02192009_(—)12378993.txt, createdFeb. 19, 2009, which is 38 KB in size. The information in the textformat of the Sequence Listing is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to specific binding agents that recognizeand bind to angiopoietins-1 (Ang-1) and/or angiopoetin-2 (Ang-2). Morespecifically, the invention relates to the production, diagnostic use,and therapeutic use of monoclonal and polyclonal antibodies, and theantigen-binding fragments thereof, which specifically bind Ang-1 and/orAng-2. Aspects of the invention also relate to hybridomas or other celllines expressing such antibodies. The described antibodies are usefulfor diagnostics and for the treatment of diseases associated with theactivity and overproduction of Ang-1 or Ang-2.

BACKGROUND OF THE INVENTION

Angiogenesis, the formation of new blood vessels from existing ones, isessential to many physiological and pathological processes. Normally,angiogenesis is tightly regulated by pro- and anti-angiogenic factors,but in the case of diseases such as cancer, ocular neovascular diseases,arthritis, and psoriasis, the process can go awry. Folkman, J., Nat.Med., 1:27-31 (1995).

There are a number of diseases known to be associated with deregulatedor undesired angiogenesis. Such diseases include, but are not limitedto, ocular neovascularisation, such as retinopathies, including diabeticretinopathy, age-related macular degeneration, psoriasis,hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease,such as a rheumatoid or rheumatic inflammatory disease, especiallyarthritis (including rheumatoid arthritis), or other chronicinflammatory disorders, such as chronic asthma, arterial orpost-transplantational atherosclerosis, endometriosis, and neoplasticdiseases, for example so-called solid tumors and liquid (orhematopoietic) tumors (such as leukemias and lymphomas). Other diseasesassociated with undesired angiogenesis will be apparent to those skilledin the art.

Although many signal transduction systems have been implicated in theregulation of angiogenesis, one of the best-characterized and mostendothelial cell-selective systems involves the Tie-2 receptor tyrosinekinase (referred to as “Tie-2” or “Tie-2R” (also referred to as “ORK”);murine Tie-2 is also referred to as “tek”) and its ligands, theangiopoietins (Gale, N. W. and Yancopoulos, G. D., Genes Dev.13:1055-1066 [1999]). There are 4 known angiopoietins; angiopoietin-1(“Ang-1”) through angiopoietin-4 (“Ang-4”). These angiopoietins are alsoreferred to as “Tie-2 ligands”. (Davis, S., et al., Cell, 87:1161-1169[1996]; Grosios, K., et al., Cytogenet Cell Genet, 84:118-120 [1999];Holash, J., et al., Investigative Ophthalmology & Visual Science,42:1617-1625 [1999]; Koblizek, T. I., et al., Current Biology, 8:529-532[1998]; Lin, P., et al., Proc Natl Acad Sci USA, 95:8829-8834 [1998];Maisonpierre, P. C., et al., Science, 277:55-60 [1997]; Papapetropoulos,A., et al., Lab Invest, 79:213-223 [1999]; Sato, T. N., et al., Nature,375:70-74 [1998]; Shyu, K. G., et al., Circulation, 98:2081-2087 [1998];Suri, C., et al., Cell, 87:1171-1180 [1996]; Suri, C., et al., Science,282:468-471 [1998]; Valenzuela, D. M., et al., Proceedings of theNational Academy of Sciences of the USA, 96:1904-1909 [1999];Witzenbichler, B., et al., J Biol Chem, 273:18514-18521 [1998]). WhereasAng-1 binding to Tie-2 stimulates receptor phosphorylation in culturedendothelial cells, Ang-2 has been observed to both agonize andantagonize Tie-2 receptor phosphorylation (Davis, S., et al., [1996],supra; Maisonpierre, P. C., et al., [1997], supra; Kim, I, J. H. Kim, etal., Oncogene 19(39): 4549-4552 (2000); Teichert-Kuliszewska, K., P. C.Maisonpierre, et al., Cardiovascular Research 49(3): 659-70 (2001)).

The phenotypes of mouse Tie-2 and Ang-1 knockouts are similar andsuggest that Ang-1-stimulated Tie-2 phosphorylation mediates remodelingand stabilization of developing vessels in utero through maintenance ofendothelial cell-support cell adhesion (Dumont, D. J., et al., Genes &Development, 8:1897-1909 [1994]; Sato, T. N., et al., Nature, 376:70-74[1995]; Sun, C., et al., [1996], supra). The role of Ang-1 in vesselstabilization is thought to be conserved in the adult, where it isexpressed widely and constitutively (Hanahan, D., Science, 277:48-50[1997]; Zagzag, D., et al., Experimental Neurology, 159:391-400 [1999]).In contrast, Ang-2 expression is primarily limited to sites of vascularremodeling, where it is thought to block Ang-1 function, therebyinducing a state of vascular plasticity conducive to angiogenesis(Hanahan, D., [1997], supra; Holash, J., et al., Science, 284:1994-1998[1999]; Maisonpierre, P. C., et al., [1997], supra).

Numerous published studies have purportedly demonstratedvessel-selective Ang-2 expression in disease states associated withangiogenesis. These pathological conditions include, for example,psoriasis, macular degeneration, and cancer (Bunone, G., et al.,American Journal of Pathology, 155:1967-1976 [1999]; Etoh, T., et al.,Cancer Research, 61:2145-2153 [2001]; Hangai, M., et al., InvestigativeOphthalmology & Visual Science, 42:1617-1625 [2001]; Holash, J., et al.,[1999] supra; Kuroda, K., et al., Journal of Investigative Dermatology,116:713-720 [2001]; Otani, A., et al., Investigative Ophthalmology &Visual Science, 40:1912-1920 [1999]; Stratmann, A., et al., AmericanJournal of Pathology, 153:1459-1466 [1998]; Tanaka, S., et al., J ClinInvest, 103:34-345 [1999]; Yoshida, Y., et al., International Journal ofOncology, 15:1221-1225 [1999]; Yuan, K., et al., Journal of PeriodontalResearch, 35:165-171 [2000]; Zagzag, D., et al., supra). Most of thesestudies have focused on cancer, in which many tumor types appear todisplay vascular Ang-2 expression. In contrast with its expression inpathological angiogenesis, Ang-2 expression in normal tissues isextremely limited (Maisonpierre, P. C., et al., [1997], supra; Mezquita,J., et al., Biochemical and Biophysical Research Communications,260:492-498 [1999]). In the normal adult, the three main sites ofangiogenesis are the ovary, placenta, and uterus; these are the primarytissues in normal (i.e., non-cancerous) tissues in which Ang-2 mRNA hasbeen detected.

Certain functional studies suggest that Ang-2 may be involved in tumorangiogenesis. Ahmad et al. (Cancer Res., 61:1255-1259 [2001]) describeAng-2 over-expression and show that it is purportedly associated with anincrease in tumor growth in a mouse xenograft model. See also Etoh etal., supra, and Tanaka et al., supra, wherein data is presentedpurportedly associating Ang-2 over expression with tumorhypervascularity. However, in contrast, Yu et al. (Am. J. Path.,158:563-570 [2001]) report data to show that overexpression of Ang-2 inLewis lung carcinoma and TA3 mammary carcinoma cells purportedlyprolonged the survival of mice injected with the correspondingtransfectants.

In the past few years, various publications have suggested Ang-1, Ang-2and Tie-2 as a possible target for anti-cancer therapy. For example,U.S. Pat. Nos. 6,166,185, 5,650,490, and 5,814,464 each disclose theconcept of anti-Tie-2 ligand antibodies and receptor bodies. U.S. PatentApp. Pub. No. 2003/0124129A1 describes certain anti-Ang 2 antibodies andtheir use in treatment of cancer. Lin et al. (Proc. Natl. Acad. Sci.USA, 95:8829-8834 [1998]) injected an adenovirus expressing solubleTie-2 into mice; the soluble Tie-2 purportedly decreased the number andsize of the tumors developed by the mice. In a related study, Lin et al.(J. Clin. Invest., 100:2072-2078 [1997]) injected a soluble form ofTie-2 into rats; this compound purportedly reduced tumor size in therats. Siemeister et al. (Cancer Res., 59:3185-3189 [1999]) generatedhuman melanoma cell lines expressing the extracellular domain of Tie-2,injected these cell lines into nude mice, and concluded that solubleTie-2 purportedly resulted in a “significant inhibition” of tumor growthand tumor angiogenesis.

Hence, an effective anti-Ang-2 therapy might benefit a vast populationof cancer patients because most solid tumors require neovascularizationto grow beyond 1-2 millimeters in diameter. Such therapy might havewider application in other angiogenesis-associated diseases as well,such as retinopathies, arthritis, and psoriasis.

SUMMARY OF THE INVENTION

Although much evidence points to the usefulness of inhibiting Ang2levels in treatment of unwanted angiogenesis (or any subset ofconditions involving unwanted generation of blood vessels, likearteriogenesis), the present state of the art does not make clearwhether the simultaneous inhibition of Ang1 would be beneficial in suchtherapies and if so what degree of Ang1 inhibition, in addition to Ang2inhibition, might prove to provide at least an additive therapeuticeffect. Accordingly, the present invention addresses an unrecognizedneed to identify new agents that specifically recognize and bind bothAng-1 and Ang-2 ligands. The binding agents, such as the antibodies ofthe present invention, have the desired activity levels in inhibitingAng2 as well as Ang1 that make them particularly useful in a variety ofsettings such as diagnostic screening, bioassays, and therapeuticintervention in diseases that are associated with Ang-1 and/or Ang-2activity, such as cancer, inflammation, and other diseases related toundesired angiogenesis.

The various embodiments of the invention relate to targeted bindingagents that specifically bind to Ang-1 and/or Ang-2 and therein inhibitphysiological or pathological angiogenesis. Mechanisms by which this canbe achieved can include, but are not limited to, either inhibition ofbinding of Ang-1 and/or Ang-2 to the Tie1 and/or Tie2 receptor,inhibition of Ang-1 and/or Ang-2 induced Tie1 and/or Tie2 signaling, orincreased clearance of Ang1 and/or Ang-2 from a patient's body, thereinreducing the effective concentration of Ang-1 and/or Ang-2.

One embodiment of the invention, the specific binding agent is a fullyhuman antibody that specifically binds to Ang-1 and/or Ang-2 andprevents Ang-1 and/or Ang-2 binding to Tie1 and/or Tie2 receptors. Yetanother embodiment of the invention is a fully human monoclonal antibodythat binds to Ang-1 and/or Ang-2 and also inhibits Ang-1 and/or Ang-2induced Tie1 and/or Tie2 phosphorylation. The antibody may bind Ang-1and/or Ang-2 with a Kd of less than about 100 pM, 30 pM, 20 pM, 10 pM, 5pM or 1 pM. Certain embodiments of the invention are antibodies of theIgG type, e.g., IgG1, IgG2, IgG3, and IgG4.

Another embodiment of the invention provides a binding agent such as anantibody comprising a heavy chain and a light chain, wherein said heavychain comprises a heavy chain variable region selected from the groupconsisting of H2 (SEQ ID NO. 1); H3 (SEQ ID NO. 2); H4(SEQ ID NO. 3); H6(SEQ ID NO. 4); H10(SEQ ID NO. 5); H11 (SEQ ID NO. 6); H5P (SEQ ID NO.7); and antigen binding fragments thereof; and said light chaincomprises a light chain variable region selected from the groupconsisting of: L1 (SEQ ID NO. 8); L2 (SEQ ID NO. 9); L4 (SEQ ID NO. 10);L6 (SEQ ID NO. 11); L7 (SEQ ID NO. 12); L8 (SEQ ID NO. 13); L9 (SEQ IDNO. 14); L11 (SEQ ID NO. 15); L12 (SEQ ID NO. 16); L13 (SEQ ID NO. 17);and antigen binding fragments thereof.

The invention also provides a specific binding agent comprising at leastone peptide selected from the group consisting of: H2 (SEQ ID NO. 1); H3(SEQ ID NO. 2); H4(SEQ ID NO. 3); H6 (SEQ ID NO. 4); H10(SEQ ID NO. 5);H11 (SEQ ID NO. 6); H5P (SEQ ID NO. 7); L1 (SEQ ID NO. 8); L2 (SEQ IDNO. 9); L4 (SEQ ID NO. 10); L6 (SEQ ID NO. 11); L7 (SEQ ID NO. 12); L8(SEQ ID NO. 13); L9 (SEQ ID NO. 14); L11 (SEQ ID NO. 15); L12 (SEQ IDNO. 16); L13 (SEQ ID NO. 17); and antigen binding fragments thereof.

It will be appreciated that the specific binding agent can be, forexample, an antibody, such as a polyclonal, monoclonal, chimeric,humanized, or a fully human antibody. The antibody may also be a singlechain antibody. Other examples of specific binding agents includepeptibodies, such as peptibody mL4-3, avimers, other forms of peptidemolecules (such as Fc-fusion molecules and Ab-fusion molecules (seeCovX-Pfizer technology)) that contain peptide sequences which recognizeand bind to a protein target (in this context, Ang2 and or Ang1ligand(s)), etc.

A specific embodiment of the invention relates to peptibodies such asmL4-3 that bind Ang1. Other embodiments of the invention include thepeptide portion of mL4-3 as well as similar Ang1-binding peptides thatcan be made by addition, deletion, and/or insertion of amino acids toand from this peptide. Similar additions, deletions, or insertions canbe made to the Fc portion of the mL4-3 peptibody. Further alterations tothe mL4-3 and peptibodies in general are well-known in the art andtaught in, for example, WO00/24782 and WO03/057134 which areincorporated herein by reference to the sections which describe andteach making binding agents that contain a randomly generated peptidewhich binds a desired target.

The invention further relates to a hybridoma that produces a monoclonalantibody according to the invention, as well as a cell lines containing(through any means such as by transfection, transformation,electroporation) with the nucleic acid sequences necessary to expressthe present specific binding agents such as the antibodies describedherein.

It will also be appreciated that the invention relates to conjugates asdescribed herein. The conjugate can be, for example, a specific bindingagent (such as an antibody) of the invention conjugated to otherproteinatious, carbohydrate, lipid, or mixed moiety molecule(s).

The invention further relates to nucleic acid molecules encoding thespecific binding agents (such as an antibody) of the invention, as wellas a vector comprising such nucleic acid molecule, as well as a hostcell containing the vector.

Additionally, the invention provides a method of making a specificbinding agent comprising, (a) transforming a host cell with at least onenucleic acid molecule encoding the specific binding agent; (b)expressing the nucleic acid molecule in said host cell; and (c)isolating said specific binding agent. The invention further provides amethod of making an antibody comprising: (a) transforming a host cellwith at least one nucleic acid molecule encoding the antibody accordingto the invention; (b) expressing the nucleic acid molecule in said hostcell; and (c) isolating said specific binding agent.

Further, the invention relates to a method of inhibiting undesiredangiogenesis in a mammal by administering a therapeutically effectiveamount of a specific binding agent according to the invention. Theinvention also provides a method of treating cancer in a mammal byadministering a therapeutically effective amount of a specific bindingagent according to the invention.

The invention also relates to a method of inhibiting undesiredangiogenesis in a mammal comprising by administering a therapeuticallyeffective amount of an antibody according to the invention. Theinvention additionally provides a method of treating cancer in a mammalcomprising administering a therapeutically effective amount of antibodyaccording to the invention.

It will be appreciated that the invention further relates topharmaceutical compositions comprising the specific binding agentaccording to the invention and a pharmaceutically acceptable formulationagent. The pharmaceutical composition may comprise an antibody accordingto the invention and a pharmaceutically acceptable formulation agent.

The invention provides a method of modulating or inhibitingangiopoietin-2 activity by administering one or more specific bindingagents of the invention. The invention also provides a method ofmodulating or inhibiting angiopoietin-2 activity by administering anantibody of the invention.

The invention further relates to a method of modulating at least one ofvascular permeability or plasma leakage in a mammal comprisingadministering a therapeutically effective amount of the specific bindingagent according to the invention. The invention also relates to a methodof treating at least one of ocular neovascular disease, obesity,hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease,inflammatory disorders, atherosclerosis, endometriosis, neoplasticdisease, bone-related disease, or psoriasis in a mammal comprisingadministering a therapeutically effective amount of a specific bindingagent according to the invention.

The invention further provides a method of modulating at least one ofvascular permeability or plasma leakage in a mammal comprisingadministering a therapeutically effective amount of an antibodyaccording to the invention. The invention also relates to a method oftreating at least one of ocular neovascular disease, obesity,hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease,inflammatory disorders, atherosclerosis, endometriosis, neoplasticdisease, bone-related disease, or psoriasis in a mammal comprisingadministering a therapeutically effective amount of an antibodyaccording to the invention.

Furthermore, the invention relates to a method of treating cancer in amammal comprising administering a therapeutically effective amount of aspecific binding agent according to the invention and a chemotherapeuticagent. It will be appreciated by those in the art that the specificbinding agent and chemotherapeutic agent need not be administeredsimultaneously.

The invention also provides a specific binding agent comprising heavychain complementarity determining region 1 (CDR 1) of any of: SEQ ID NO.18; The invention further relates to a specific binding agent comprisingheavy chain complementarity determining region 2 (CDR 2) of any of: SEQID NO. 26; SEQ ID NO. 27; SEQ ID NO. 28; SEQ ID NO. 29; and antigenbinding fragments thereof.

The invention also relates to a specific binding agent comprising heavychain complementarity determining region 3 (CDR 3) of any of: SEQ ID NO.32; SEQ ID NO. 34; SEQ ID NO. 35; SEQ ID NO. 37; SEQ ID NO. 38; SEQ IDNO. 39); and antigen binding fragments thereof.

The invention also provides a specific binding agent comprising lightchain complementarity determining region 1 (CDR 1) of any of: SEQ ID NO.19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID NO. 23; SEQ IDNO. 24; SEQ ID NO. 25; and antigen binding fragments thereof;

The invention further relates to a specific binding agent comprisinglight chain complementarity determining region 2 (CDR 2) of any of: SEQID NO. 27; SEQ ID NO. 30; SEQ ID NO. 31; and antigen binding fragmentsthereof.

The invention also relates to a specific binding agent comprising lightchain complementarity determining region 3 (CDR 3) of any of: SEQ IDNO.33; SEQ ID NO. 36; SEQ ID NO. 40; and antigen binding fragmentsthereof.

Other embodiments of the invention include isolated nucleic acidmolecules encoding any of the antibodies described herein, vectorshaving isolated nucleic acid molecules encoding anti-Ang-1 and/orAnti-Ang-2 antibodies or a host cell transformed with any of suchnucleic acid molecules. In addition, one embodiment of the invention isa method of producing an anti-Ang-1 and/or anti-Ang-2 antibody byculturing host cells under conditions wherein a nucleic acid molecule isexpressed to produce the antibody followed by recovering the antibody.It should be realized that embodiments of the invention also include anynucleic acid molecule which encodes an antibody or fragment of anantibody of the invention including nucleic acid sequences optimized forincreasing yields of antibodies or fragments thereof when transfectedinto host cells for antibody production.

A further embodiment herein includes a method of producing high affinityantibodies to Ang-1 and/or Ang-2 by immunizing a mammal with human Ang-1or 2, or a fragment thereof, and one or more orthologous sequences orfragments thereof.

Moreover, the invention relates to a method of detecting the level ofAng-1 or Ang-2 in a biological sample by (a) contacting a specificbinding agent of the invention with the sample; and (b) determining theextent of binding of the specific binding agent to the sample. Theinvention also relates to a method of detecting the level of Ang-2 in abiological sample by (a) contacting an antibody of the invention withthe sample; and (b) determining the extent of binding of the antibody tothe sample.

The invention also relates to a method of inhibiting undesiredangiogenesis in a mammal comprising administering a therapeuticallyeffective amount of a polypeptide or composition as described herein.The invention also relates to a method of modulating angiogenesis in amammal comprising administering a therapeutically effective amount of apolypeptide or composition as described herein. The invention furtherrelates to a method of inhibiting tumor growth characterized byundesired angiogenesis in a mammal comprising administering atherapeutically effective amount of a polypeptide or composition asdescribed herein. Additionally, the invention relates to a method oftreating cancer in a mammal comprising administering a therapeuticallyeffective amount of a polypeptide or composition as described herein,and a chemotherapeutic agent. The specific polypeptide or composition asdescribed herein and chemotherapeutic agent need not be administeredsimultaneously. In a preferred embodiment, the chemotherapeutic agent isat least one of 5-FU, CPT-11, and Taxotere. It will be appreciated,however, that other suitable chemotherapeutic agents and other cancertherapies can be used.

Additionally, the invention relates to a method of treating cancer in amammal comprising administering a therapeutically effective amount of apolypeptide or composition as described herein, and an anti-VEGF agentor a multikinase inhibitor (MKI). In a preferred embodiment, theanti-VEGF agent or a multikinase inhibitor (MKI) would be chosen fromAvastin® (bevacizumab), Lucentis® (ranibizumab), Macugen® (pegaptanib),Sutent® (sunitinib), Nexavar® (sorafenib), motesanib diphosphate,Zactima® (vandetanib), Recentin (AZD 2171), AG-013736 (axitinib). Itwill be appreciated, however, that other suitable anti-angiogenic agentsand other cancer therapies can be used.

It will be appreciated that the specific binding agents of the inventionare used to treat a number of diseases associated with deregulated orundesired angiogenesis. Such diseases include, but are not limited to,ocular neovascularisation, such as retinopathies (including diabeticretinopathy and age-related macular degeneration) psoriasis,hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease,such as a rheumatoid or rheumatic inflammatory disease, especiallyarthritis (including rheumatoid arthritis), or other chronicinflammatory disorders, such as chronic asthma, arterial orpost-transplantational atherosclerosis, endometriosis, and neoplasticdiseases, for example so-called solid tumors and liquid tumors (such asleukemias). Additional diseases which can be treated by administrationof the specific binding agents will be apparent to those skilled in theart. Such additional diseases include, but are not limited to, obesity,vascular permeability, plasma leakage, and bone-related disorders,including osteoporosis. Thus, the invention further relates to methodsof treating these diseases associated with deregulated or undesiredangiogenesis.

Additional embodiments of the invention include a specific binding agentcomprising at least one peptide selected from the group consisting of:SEQ ID NO. 1; SEQ ID NO. 2; SEQ ID NO. 3; SEQ ID NO. 4; SEQ ID NO. 5;SEQ ID NO. 6; SEQ ID NO. 7; SEQ ID NO. 8; SEQ ID NO. 9; SEQ ID NO. 10;SEQ ID NO. 11; SEQ ID NO. 12; SEQ ID NO. 13; SEQ ID NO. 14; SEQ ID NO.15; SEQ ID NO. 16; SEQ ID NO. 17; and antigen-binding fragments thereof.Also contemplated are antibodies containing the aforementionedpolypeptide sequences. These antibodies are polyclonal, monoclonal,chimeric, humanized, or fully human antibodies. They are single chainantibody as well as multi-chain antibodies. Hybridomas that produce themonoclonal antibodies are also contemplated, as well as, nucleic acidmolecules encoding the polypeptides and the antibodies, the vectorscontaining these nucleic acid molecules, and the host cells, such as CHOcells, that contain and express them. A method of making a binding agentor an antibody of the present invention comprises transforming a hostcell with at least one nucleic acid molecule encoding the binding agentor antibody; expressing the nucleic acid molecule in said host cell; andisolating said specific binding agent or antibody.

A diagnostic use of the invention includes a method of detecting thelevel of angiopoietin-1 and/or angiopoietin-in a biological samplecomprising contacting an antibody or binding agent described herein withsaid biological sample; and determining the extent of binding of theantibody or binding agent to said sample.

Amongst the specific therapeutic uses of the invention are methods ofinhibiting undesired angiogenesis (or any subset of conditions involvingunwanted generation of blood vessels, like arteriogenesis), in a mammalcomprising administering a therapeutically effective amount of theisolated polypeptides or the binding agents such as antibodies madetherefrom. Amongst such undesired angiogenesis (or any subset ofconditions involving unwanted generation of blood vessels, likearteriogenesis), are cancer and inflammatory diseases in mammals.Therefore, a pharmaceutical composition is contemplated that comprisesthe isolated polypeptide, binding agent or antibody of the invention inadmixture with a pharmaceutical carrier therefore. Pharmaceuticallyacceptable formulation agents, of course, are often used to prepare suchpharmaceutical compositions for administration to subjects in needthereof.

Other methods of using the compositions of the present invention includea method of modulating or inhibiting angiopoietin-1 and/orangiopoietin-2 activity comprising administering to a patient theisolated polypeptide, binding agent or antibody described herein. Suchmethods of modulating or inhibiting angiopoietin-1 and/or angiopoietin-2activity comprise administering to a patient the polypeptide, bindingagent, or antibody described herein. Such methods include modulating atleast one of vascular permeability or plasma leakage in a mammalcomprising administering to a mammal a therapeutically effective amountof the isolated polypeptide, binding agent or antibody described herein.Also included are methods of treating at least one of ocular neovasculardisease, obesity, hemangioblastoma, hemangioma, arteriosclerosis,inflammatory disease, inflammatory disorders, atherosclerosis,endometriosis, neoplastic disease, bone-related disease, or psoriasis.

Also contemplated is a combotherapy (combination therapy) method such asa method of treating cancer in a mammal comprising administering atherapeutically effective amount of an isolated polypeptide, bindingagent or antibody described herein and a chemotherapeutic agent. In suchmethods, sometimes the isolated polypeptide, binding agent or antibodyand the chemotherapeutic agent are administered simultaneously and atother times are not, depending upon the specific condition, regulatoryapproval, and the judgement of the medical professionals.

Other types of combotherapy include a method of treating cancer in amammal comprising administering to a subject in need thereof atherapeutically effective amount of an isolated polypeptide, bindingagent or antibody described herein and a second molecule that binds aligand to any one of the VEGF receptors 1-3. Examples of such secondmolecules that bind a ligand to any one of the VEGF receptors 1-3 areAvastin®, Lucentis®, and Macugen®.

Use of the polypeptides, binding agents, or antibodies described hereinare also contemplated in combination with small molecule agents fortherapeutic administration to subjects in need thereof. Such smallmolecule agents include those that modulate the signaling of any one ofthe VEGF receptors 1-3 as well as those that are multikinase inhibitors.For example, Sutent®, Nexavar®, Motesanib diphosphate, Axitinib,Zactima, AZD 2171, Recentin, and AG-013736 are contemplated for use incombotherapy with the polypepetides, binding agents, and antibodiesdescribed herein.

Certain other embodiments of the invention relate to a specific bindingagent comprising CDR 1 of any of SEQ ID NO. 18; SEQ ID NO. 19; SEQ IDNO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID NO. 23; SEQ ID NO. 24; SEQID NO. 25; a specific binding agent comprising CDR 2 of any of SEQ IDNO. 26; SEQ ID NO. 27; SEQ ID NO. 28; SEQ ID NO. 29; SEQ ID NO. 30; SEQID NO. 31; and a specific binding agent comprising CDR 3 of any of SEQID NO. 32; SEQ ID NO. 33; SEQ ID NO. 34; SEQ ID NO. 35; SEQ ID NO. 36;SEQ ID NO. 37; SEQ ID NO. 38; SEQ ID NO. 39; SEQ ID NO. 40. The specificbinding agent may comprise 1, 2, 3, 4, 5, or 6 CDRs.

Similarly, nucleic acid molecules encoding the above-mentioned specificbinding agents are contemplated. Also contemplated is a method ofdetecting the level of angiopoietin-1 and/or angiopoietin-2 in abiological sample comprising contacting a specific binding agent asdescribed herein with said biological sample; and determining the extentof binding of the specific binding agent to said sample. Additionally, amethod is contemplated for detecting the level of angiopoietin-1 and/orangiopoietin-2 in a biological sample comprising contacting any one ofthe antibodies described herein with said biological sample; anddetermining the extent of binding of the antibody to said sample.

A further embodiment of the invention is an antibody comprising a heavychain and a light chain, the heavy chain comprising a heavy chainvariable region selected from the group consisting of SEQ ID NO. 1; SEQID NO. 2; SEQ ID NO. 3; SEQ ID NO. 4; SEQ ID NO. 5; SEQ ID NO. 6 and,SEQ ID NO. 7; and the light chain comprising a light chain variableregion selected from the group consisting of SEQ ID NO. 8; SEQ ID NO. 9;SEQ ID NO. 10; SEQ ID NO. 11; SEQ ID NO. 12; SEQ ID NO. 13; SEQ ID NO.14; SEQ ID NO. 15; SEQ ID NO. 16 and, SEQ ID NO. 17; as well as antigenbinding fragments thereof. Naturally, nucleic acid molecules encodingthe above-described antibodies and antigen-binding fragments are alsocontemplated.

In another embodiment, the present invention is directed to an isolatedantibody comprising a heavy chain and a light chain, the light chaincomprising a light chain variable domain and the heavy chain comprisinga heavy chain variable domain, the heavy chain variable domain havingthe sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQID NO: 7; wherein the antibody specifically binds to at least one ofAng1 and Ang2 ligands of Tie 2 receptor.

In a further embodiment, the invention is an isolated antibodycomprising a heavy chain and a light chain, the heavy chain comprising aheavy chain variable domain and the light chain comprising a light chainvariable domain, the light chain variable domain having the sequenceselected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; wherein the antibodyspecifically binds to at least one of Ang1 and Ang2 ligands of Tie 2receptor.

In an additional embodiment, the invention is directed to an isolatedantibody comprising a heavy chain and light chain, the heavy chaincomprising a heavy chain variable domain and the light chain comprisinga light chain variable domain, wherein the heavy chain variable domaincomprises 1, 2, or 3 heavy chain CDRs selected from the group of HC CDRsconsisting of SEQ ID NOs: 18, 26, 28, 32, 34, 35, 37, 38 and, 39, andwherein the antibody specifically binds to at least one of Ang1 and Ang2ligands of Tie 2 receptor.

In another embodiment, the invention is directed to an isolated antibodywhich comprises a light chain and a heavy chain, wherein the light chaincomprises a light chain variable domain and the heavy chain comprises aheavy chain variable domain, wherein the light chain variable domaincomprises 1, 2, or 3, light chain CDRs selected from the group of LCCDRs consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 27, 33, 36, 40, andwherein the antibody specifically binds to at least one of Ang1 and Ang2ligands of Tie 2 receptor.

In a further embodiment, the invention is an isolated antibody whichcomprises a heavy chain and a light chain, wherein the heavy chaincomprises a heavy chain variable domain and the light chain comprises alight chain variable domain, wherein the heavy chain comprises 3 heavychain (HC) CDRs and said light chain variable domain comprises 3 lightchain (LC) CDRs, wherein the sequences of said HC and LC CDRs of theantibody are selected from the group consisting of:

(a) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 19, 27, 33 of theLC,

(b) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 19, 27, 33 of theLC,

(c) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20, 27, 36 of theLC,

(d) SEQ ID NOs: 18, 26, 37 of the HC plus SEQ ID NOs: 19, 27, 33 of theLC,

(e) SEQ ID NOs: 18, 26, 38 of the HC plus SEQ ID NOs: 19, 27, 33 of theLC,

(f) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 19, 27, 33 of theLC,

(g) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 21, 27, 33 of theLC,

(h) SEQ ID NOs: 18, 28, 39 of the HC plus SEQ ID NOs: 19, 27, 33 of theLC,

(i) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 22, 27, 33 of theLC,

(j) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 22, 27, 33 of theLC,

(k) SEQ ID NOs: 18, 29, 39 of the HC plus SEQ ID NOs: 19, 27, 33 of theLC,

(l) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 23, 27, 33 of theLC,

(m) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20, 27, 40 of theLC,

(n) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 21, 27, 33 of theLC,

(O) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 24, 27, 33 of theLC,

(p) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 21, 27, 33 of theLC,

(q) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 23, 27, 33 of theLC,

(r) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20, 30, 33 of theLC,

(s) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 25, 27, 33 of theLC,

(t) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20, 30, 33 of theLC,

(u) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20, 27, 40 of theLC, and

(v) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20, 31, 33 of theLC;

wherein the antibody specifically binds to at least one of Ang1 and Ang2ligands of Tie 2 receptor.

The present invention also is directed to an antibody having a heavychain and light chain, where the light chain has a light chain variabledomain having three LC CDRs of any one of (a) through (v), supra,wherein the antibody specifically to at least one of Ang1 and Ang2ligands of Tie 2 receptor.

Additionally, the present invention also is directed to an antibodyhaving a heavy chain and light chain, where the heavy chain has a heavychain variable domain having three HC CDRs of any one of (a) through(v), supra, wherein the antibody specifically to at least one of Ang1and Ang2 ligands of Tie 2 receptor.

Nucleic acid molecules encoding any of the aforementioned antibodies andantigen-binding fragments thereof are also contemplated. Otherembodiments of this invention will be readily apparent from thedisclosure provided herewith.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph of tumor size (y-axis) versus time (x-axis) intumor-bearing mice treated with either an anti-Ang1/2 antibody (H4L4,H4L11, or H6L7) of the invention or a highly potent control peptibody(AMG 386) or antibody 536, compared to treatment with an isotype controlantibody. Details are described in the Examples.

FIG. 2 depicts the tumor burden (% viable tumor [bisected section] xtumor weight) in tumor-bearing mice treated with an anti-Ang1/2 antibody(H4L4, H4L11, or H6L7) of the invention or a highly potent controlpeptibody (AMG 386) or antibody 536 compared to treatment with anisotype control antibody. Details are described in the Examples.

FIG. 3 depicts the effect of H4L4, H4L11, and H6L7 of the invention, ahighly potent control peptibody (AMG 386) and antibody 536 onendothelial cell proliferation in Colo205 tumor-bearing mice. Detailsare described in the Examples.

FIG. 4 depicts the H4L4 antibody dose-response relationship in Colo205tumor-bearing mice. Details are described in the Examples.

FIG. 5 depicts the effect of H4L4 antibody on Colo205 tumor burden invivo. Details are described in the Examples.

FIG. 6 depicts the effect of the antibody H4L4 on endothelial cellproliferation in Colo205 tumor-bearing mice. Details are described inthe Examples.

FIG. 7 depicts systemically administered mL4-3 neutralizes Ang1-inducedTie2 phosphorylation in mouse lungs. Mice (n=3 per group) were treatedwith L1-7(N) (2 mg/kg), mL4-3 (20 mg/kg) or Fc control (20 mg/kg) dailyfor 23 days prior to i.v. challenge with Ang1 or BSA. Mouse lungs weresubsequently harvested, and the levels of phosphorylated Tie2 weredetermined by immunoprecipitation-Western blot analysis. Data are meanvalues±SE. *P=0.0005 vs Ang1 plus Fc, ANOVA with Fisher's post hoc test.

FIG. 8 depicts pharmacologic inhibition of Ang1 during earlyorganogenesis alters heart development. A) Mouse embryos exposed to 300mg/kg mL4-3 (right panel) had smaller hearts with fewer, narrower, andmore widely spaced trabeculae relative to the larger hearts with large,wide trabeculae found in stage-matched embryos exposed to 300 mg/kg Fccontrol (left panel). Representative images are shown. B) Incidence ofcardiac abnormalities in Fc- and mL4-3-treated embryos. *P<0.0001 vs Fc,chi-square test.

FIG. 9 depicts the effect of combined Ang1 and Ang2 inhibition on thegrowth of Colo205 tumor xenografts. Mice (n=10 per group) were implantedwith Colo205 cells, and treatment began when tumors reachedapproximately 500 mm³ with Fc control (5.2 mg/kg QD), mL4-3 (3.2 mg/kgQD), L1-7(N) (2.0 mg/kg QD), L1-7(N) combined with mL4-3 (at the samedosing regimens used in the single-agent groups), or AMG 386 (5.6 mg/kgtwice per week). One of four representative experiments is shown. Dataare mean values±SE. *P<0.0001 vs L1-7(N), RMANOVA with Scheffé post hoctest.

FIG. 10 depicts the effect of Ang1 and Ang2 antagonism on tumorendothelial cell proliferation, corneal angiogenesis, and retinalangiogenesis. A) The effect of inhibition of Ang1 and Ang2 on BrdUuptake in mouse endothelial cells derived from Colo205 tumor xenografts.Tumor-bearing mice were treated for 3 days with Fc (5.7 mg/kg QD), AMG386 (6 mg/kg single dose), L1-7(N) (2.2 mg/kg QD), mL4-3 (3.5 mg/kg QD),or L1-7(N) combined with mL4-3 (at the same doses and schedules used inthe single-agent groups). Each bar represents mean endothelial:totalmouse cell BrdU ratios (n=3). Data are mean values±SE. *P<0.05 vs. Fc,unpaired Student's t-test. B and C) The effect of inhibition of Ang1 andAng2 on (B) VEGF-induced and (C) bFGF-induced corneal angiogenesis.Angiogenesis was induced by implanting VEGF- or bFGF-soaked nylon discsinto the corneal stroma of rats (n=8 per group). Treatment was initiatedone day prior to corneal implantation and continued every 3 days with:Fc (60 mg/kg), L1-7(N) (5 mg/kg), mL4-3 (60 mg/kg) and L1-7(N) combinedwith mL4-3 (at the same dose and schedule used in the single-agentgroups). Data are mean values±SE. ^(†)P<0.0001 vs Fc+VEGF (B);^(#)P<0.002 vs Fc+bFGF (C), ANOVA with Fisher's post hoc test. D)Inhibition of Ang2 prevents oxygen-induced neovascularization in themouse retina. Starting on postnatal day P8, pups (n=5 per group) weretreated daily s.c. for nine days with Fc (200 mg/kg) L1-7(N) (100 mg/kg)mL4-3 (100 mg/kg) or L1-7(N) combined with mL4-3 (at the same dose andschedule used in the single-agent groups). Data are mean values±SE.^(§)P<0.0001 vs Fc, ANOVA with Fisher's post hoc test.

FIG. 11 depicts Ang1 and Ang2 inhibitors cooperatively suppress ovarianfollicular angiogenesis. HCG was used to induce superovulation in mice.Fc (300 mg/kg), mL4-3 (150 mg/kg), L1-7(N) (150 mg/kg), or anmL4-3/L1-7(N) combination (150 mg/kg each) administered s.c. (n=7-10mice per group) were evaluated for the ability to preventneovascularization in ovulating follicles. Blood vessel area wascalculated from anti-CD31 immunostained sections of individualfollicles. Data are mean values±SE. Two independent experiments areshown. *P=0.005 comparing mL4-3/L1-7(N) combination vs either singleagent alone; ^(#)P<0.05 vs Fc, ANOVA with Dunnett's post hoc test.

DETAILED DESCRIPTION OF INVENTION

The section headings are used herein for organizational purposes only,and are not to be construed as in any way limiting the subject matterdescribed.

Standard techniques may be used for recombinant DNA molecule, protein,and antibody production, as well as for tissue culture and celltransformation. Enzymatic reactions and purification techniques aretypically performed according to the manufacturer's specifications or ascommonly accomplished in the art using conventional procedures such asthose set forth in Sambrook et al. (Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.[1989]), or as described herein. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques may be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The terms used to describe the present invention, unless specificallydefined herein, shall have their meaning as understood and used in theart.

It should be noted that the terms H5 and H5P are used interchangeablyand refer to the heavy chain used in various embodiments of theinvention, e.g., mAbs named as H5L7, H5L6, H5L8, H5L4, H5L11, H5L1,H5L12, and H5L9.

The term “Ang-2” refers to the polypeptide set forth in FIG. 6 of U.S.Pat. No. 6,166,185 (“Tie-2 ligand-2”), incorporated herein by reference,or fragments thereof as well as related polypeptides which includeallelic variants, splice variants, derivatives, substitution, deletions,and/or insertion variants, fusion peptides and polypeptides, andinterspecies homologs. The Ang-2 polypeptide may or may not includeadditional terminal residues, e.g., leader sequences, targetingsequences, amino terminal methionine, amino terminal methionine andlysine residues, and/or tag or fusion proteins sequences, depending onthe manner in which it is prepared.

The term “specific binding agent” refers to a molecule, preferably aproteinaceous molecule, that binds Ang-2 as well as Ang-1 (and variantsand derivatives thereof as defined herein) with a greater affinity thanother angiopoietins. A specific binding agent may be a protein, peptide,nucleic acid, carbohydrate, lipid, or small molecular weight compoundwhich binds preferentially to Ang-2 and Ang-1. In a preferredembodiment, the specific binding agent according to the presentinvention is an antibody, such as a polyclonal antibody, a monoclonalantibody (mAb), a chimeric antibody, a CDR-grafted antibody, amulti-specific antibody, a bi-specific antibody, a catalytic antibody, ahumanized antibody, a human antibody, an anti-idiotypic (anti-Id)antibody, and antibodies that can be labeled in soluble or bound form,as well as antigen-binding fragments, variants or derivatives thereof,either alone or in combination with other amino acid sequences, providedby known techniques. Such techniques include, but are not limited toenzymatic cleavage, chemical cleavage, peptide synthesis or recombinanttechniques. The anti-Ang-2 and Ang-1 specific binding agents of thepresent invention are capable of binding portions of Ang-2 and Ang-1that modulate, e.g., inhibit or promote, the biological activity ofAng-2 and Ang-1 and/or other Ang-2- and Ang-1-associated activities.

The term “polyclonal antibody” refers to a heterogeneous mixture ofantibodies that recognize and bind to different epitopes on the sameantigen. Polyclonal antibodies may be obtained from crude serumpreparations or may be purified using, for example, antigen affinitychromatography, or Protein A/Protein G affinity chromatography.

The term “monoclonal antibodies” refers to a collection of antibodiesencoded by the same nucleic acid molecule that are optionally producedby a single hybridoma (or clone thereof) or other cell line, or by atransgenic mammal such that each monoclonal antibody will typicallyrecognize the same epitope on the antigen. The term “monoclonal” is notlimited to any particular method for making the antibody, nor is theterm limited to antibodies produced in a particular species, e.g.,mouse, rat, etc.

The term “chimeric antibodies” refers to antibodies in which a portionof the heavy and/or light chain is identical with or homologous to acorresponding sequence in an antibody derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is/are identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included areantigen-binding fragments of such antibodies that exhibit the desiredbiological activity (i.e., the ability to specifically bind Ang-2). See,U.S. Pat. No. 4,816,567 and Morrison et al., Proc Natl Acad Sci (USA),81:6851-6855 [1985].

The term “CDR grafted antibody” refers to an antibody in which the CDRfrom one antibody of a particular species or isotype is recombinantlyinserted into the framework of another antibody of the same or differentspecies or isotype.

The term “multi-specific antibody” refers to an antibody having variableregions that recognize more than one epitope on one or more antigens. Asubclass of this type of antibody is a “bi-specific antibody” whichrecognizes two distinct epitopes on the same or different antigens.

“Catalytic” antibodies refers to antibodies wherein one or morecytotoxic, or more generally one or more biologically active, moietiesare attached to the targeting binding agent.

The term “humanized antibody” refers to a specific type of CDR-graftedantibody in which the antibody framework region is derived from a humanbut each CDR is replaced with that derived from another species, such asa murine CDR. The term “CDR” is defined infra.

The term “fully human” antibody refers to an antibody in which both theCDR and the framework are derived from one or more human DNA molecules.

The term “anti-idiotype” antibody refers to any antibody thatspecifically binds to another antibody that recognizes an antigen.Production of anti-idiotype antibodies can be performed by any of themethods described herein for production of Ang-2-specific antibodiesexcept that these antibodies arise from e.g., immunization of an animalwith an Ang-2-specific antibody or Ang-2-binding fragment thereof,rather than Ang-2 polypeptide itself or a fragment thereof.

The term “variants,” as used herein, include those polypeptides whereinamino acid residues are inserted into, deleted from and/or substitutedinto the naturally occurring (or at least a known) amino acid sequencefor the binding agent. Variants of the invention include fusion proteinsas described below.

“Derivatives” include those binding agents that have been chemicallymodified in some manner distinct from insertion, deletion, orsubstitution variants.

“Specifically binds” refers to the ability of a specific binding agent(such as an antibody or fragment thereof) of the present invention torecognize and bind mature, full-length or partial-length targetpolypeptide (herein Ang-2 and Ang-1), or an ortholog thereof, such thatits affinity (as determined by, e.g., Affinity ELISA or BIAcore assaysas described herein) or its neutralization capability (as determined bye.g., Neutralization ELISA assays described herein, or similar assays)is at least 10 times as great, but optionally 50 times as great, 100,250 or 500 times as great, or even at least 1000 times as great as theaffinity or neutralization capability of the same for any otherangiopoietin or other peptide or polypeptide.

The term “antigen binding domain” or “antigen binding region” refers tothat portion of the specific binding agent (such as an antibodymolecule) which contains the specific binding agent amino acid residues(or other moieties) that interact with an antigen and confer on thebinding agent its specificity and affinity for the antigen. In anantibody, the antigen-binding domain is commonly referred to as the“complementarity-determining region, or CDR.”

The term “epitope” refers to that portion of any molecule capable ofbeing recognized by and bound by a specific binding agent, e.g. anantibody, at one or more of the binding agent's antigen binding regions.Epitopes usually consist of chemically active surface groupings ofmolecules, such as for example, amino acids or carbohydrate side chains,and have specific three-dimensional structural characteristics as wellas specific charge characteristics. Epitopes as used herein may becontiguous or non-contiguous. Moreover, epitopes may be mimetic in thatthey comprise a three dimensional structure that is identical to theepitope used to generate the antibody, yet comprise none or only some ofthe amino acid residues found in the Ang-2 used to stimulate theantibody immune response.

The term “inhibiting and/or neutralizing epitope” is an epitope, whichwhen bound by a specific binding agent such as an antibody, results inthe loss of (or at least the decrease in) biological activity of themolecule, cell, or organism containing such epitope, in vivo, in vitro,or in situ. In the context of the present invention, the neutralizingepitope is located on or is associated with a biologically active regionof Ang-2. Alternatively, the term “activating epitope” is an epitope,which when bound by a specific binding agent of the invention, such asan antibody, results in activation, or at least maintenance of abiologically active conformation, of Ang-2.

The term “antibody fragment” refers to a peptide or polypeptide whichcomprises less than a complete, intact antibody. Complete antibodiescomprise two functionally independent parts or fragments: an antigenbinding fragment known as “Fab,” and a carboxy terminal crystallizablefragment known as the “Fc” fragment. The Fab fragment includes the firstconstant domain from both the heavy and light chain (CH1 and CL1)together with the variable regions from both the heavy and light chainsthat bind the specific antigen. Each of the heavy and light chainvariable regions includes three complementarity determining regions(CDRs) and framework amino acid residues which separate the individualCDRs. The Fc region comprises the second and third heavy chain constantregions (CH2 and CH3) and is involved in effector functions such ascomplement activation and attack by phagocytic cells. In someantibodies, the Fc and Fab regions are separated by an antibody “hingeregion,” and depending on how the full length antibody isproteolytically cleaved, the hinge region may be associated with eitherthe Fab or Fc fragment. For example, cleavage of an antibody with theprotease papain results in the hinge region being associated with theresulting Fc fragment, while cleavage with the protease pepsin providesa fragment wherein the hinge is associated with both Fab fragmentsimultaneously. Because the two Fab fragments are in fact covalentlylinked following pepsin cleavage, the resulting fragment is termed theF(ab′)2 fragment.

An Fc domain may have a relatively long serum half-life, whereas a Fabis short-lived. [Capon et al., Nature, 337: 525-31 (1989)] Whenexpressed as part of a fusion protein, an Fc domain can impart longerhalf-life or incorporate such functions as Fc receptor binding, ProteinA binding, complement fixation and perhaps even placental transfer intothe protein to which it is fused. The Fc region may be a naturallyoccurring Fc region, or may be altered to improve certain qualities,such as therapeutic qualities or circulation time.

The term “variable region” or “variable domain” refers to a portion ofthe light and/or heavy chains of an antibody, typically includingapproximately the amino-terminal 120 to 130 amino acids in the heavychain and about 100 to 110 amino terminal amino acids in the lightchain. The variable regions typically differ extensively in amino acidsequence even among antibodies of the same species. The variable regionof an antibody typically determines the binding and specificity of eachparticular antibody for its particular antigen. The variability insequence is concentrated in those regions referred to ascomplementarity-determining regions (CDRs), while the more highlyconserved regions in the variable domain are called framework regions(FR). The CDRs of the light and heavy chains contain within them theamino acids which are largely responsible for the direct interaction ofthe antibody with antigen, however, amino acids in the FRs cansignificantly affect antigen binding/recognition as discussed hereininfra.

The term “light chain” when used in reference to an antibodycollectively refers to two distinct types, called kappa (k) or lambda(l) based on the amino acid sequence of the constant domains.

The term “heavy chain” when used in reference to an antibodycollectively refers to five distinct types, called alpha, delta,epsilon, gamma and mu, based on the amino acid sequence of the heavychain constant domain. The combination of heavy and light chains giverise to five known classes of antibodies: IgA, IgD, IgE, IgG and IgM,respectively, including four known subclasses of IgG, designated asIgG₁, IgG₂, IgG₃ and IgG₄.

The term “naturally occurring” when used in connection with biologicalmaterials such as nucleic acid molecules, polypeptides, host cells, andthe like, refers to those which are found in nature and not modified bya human being.

The term “isolated” when used in relation to Ang-2 or to a specificbinding agent of Ang-2 refers to a compound that is free from at leastone contaminating polypeptide or compound that is found in its naturalenvironment, and preferably substantially free from any othercontaminating mammalian polypeptides that would interfere with itstherapeutic or diagnostic use.

The term “mature” when used in relation to Ang-2, anti-Ang-2 antibody,or to any other proteinaceous specific binding agent of Ang-2 refers toa peptide or a polypeptide lacking a leader or signal sequence. When abinding agent of the invention is expressed, for example, in aprokaryotic host cell, the “mature” peptide or polypeptide may alsoinclude additional amino acid residues (but still lack a leadersequence) such as an amino terminal methionine, or one or moremethionine and lysine residues. A peptide or polypeptide produced inthis manner may be utilized with or without these additional amino acidresidues having been removed.

Specific Binding Agents and Antibodies

As used herein, the term “specific binding agent” refers to a moleculethat has specificity for recognizing and binding Ang-2 and Ang-1, asdescribed herein. Suitable specific binding agents include, but are notlimited to, antibodies and derivatives thereof, polypeptides, and smallmolecules. Suitable specific binding agents may be prepared usingmethods known in the art. An exemplary Ang-2 and Ang-1 polypeptidespecific binding agent of the present invention is capable of binding acertain portion of the Ang-2 and Ang-1 polypeptides, and preferablymodulating the activity or function of Ang-2 and Ang-1 polypeptides.

Specific binding agents such as antibodies and antibody fragments thatspecifically bind Ang-2 and Ang-1 polypeptides are within the scope ofthe present invention. The antibodies may be polyclonal includingmono-specific polyclonal, monoclonal (mAbs), recombinant, chimeric,humanized such as CDR-grafted, human, single chain, catalytic,multi-specific and/or bi-specific, as well as antigen-binding fragments,variants, and/or derivatives thereof.

Polyclonal antibodies against Ang2 and Ang1 polypeptides generally areproduced in animals (e.g., rabbits, hamsters, goats, sheep, horses,pigs, rats, gerbils, guinea pigs, mice, or any other suitable mammal, aswell as other non-mammal species) by means of multiple subcutaneous orintraperitoneal injections of Ang-2 and/or Ang-1 polypeptide or afragment thereof with or without an adjuvant. Such adjuvants include,but are not limited to, Freund's complete and incomplete, mineral gelssuch as aluminum hydroxide, and surface-active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are potentially useful humanadjuvants. It may be useful to conjugate an antigen polypeptide to acarrier protein that is immunogenic in the species to be immunized, suchas keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Also, aggregating agents such as alum areused to enhance the immune response. After immunization, the animals arebled and the serum is assayed for anti-Ang-2 polypeptide antibody titerwhich can be determined using the assays described herein under“Examples”. Polyclonal antibodies may be utilized in the sera from whichthey were detected, or may be purified from the sera, using, forexample, antigen affinity chromatography or Protein A or G affinitychromatography. Monoclonal antibodies directed toward Ang-2 polypeptidescan be produced using, for example but without limitation, thetraditional “hybridoma” method or the newer “phage display” technique.For example, monoclonal antibodies of the invention may be made by thehybridoma method as described in Kohler et al., Nature 256:495 [1975];the human B-cell hybridoma technique [Kosbor et al., Immunol Today 4:72(1983); Cote et al., Proc Natl Acad Sci (USA) 80: 2026-2030 (1983);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63, Marcel

Dekker, Inc., New York, (1987)] and the EBV-hybridoma technique [Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96, (1985)]. Also provided by the invention are hybridomacell lines that produce monoclonal antibodies reactive with Ang-2polypeptides.

When the hybridoma technique is employed, myeloma cell lines can beused. Such cell lines suited for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). For example, cell lines used in mousefusions are Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14,FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and 5194/5XX0 Bul; cell lines usedin rat fusions are R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other celllines useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6. Hybridomas and other cell lines that produce monoclonalantibodies are contemplated to be novel compositions of the presentinvention.

The phage display technique may also be used to generate monoclonalantibodies from any species. Preferably, this technique is used toproduce fully human monoclonal antibodies in which a polynucleotideencoding a single Fab or Fv antibody fragment is expressed on thesurface of a phage particle. [Hoogenboom et al., J Mol Biol 227: 381(1991); Marks et al., J Mol Biol 222: 581 (1991); see also U.S. Pat. No.5,885,793)]. Each phage can be “screened” using binding assays describedherein to identify those antibody fragments having affinity for Ang-2.Thus, these processes mimic immune selection through the display ofantibody fragment repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding toAng-2. One such procedure is described in PCT Application No.PCT/US98/17364, filed in the name of Adams et al., which describes theisolation of high affinity and functional agonistic antibody fragmentsfor MPL- and msk-receptors using such an approach. In this approach, acomplete repertoire of human antibody genes can be created by cloningnaturally rearranged human V genes from peripheral blood lymphocytes aspreviously described [Mullinax et al., Proc Natl Acad Sci (USA) 87:8095-8099 (1990)].

Once a polynucleotide sequences are identified which encode each chainof the full length monoclonal antibody or the Fab or Fv fragment(s) ofthe invention, host cells, either eukaryotic or prokaryotic, may be usedto express the monoclonal antibody polynucleotides using recombinanttechniques well known and routinely practiced in the art. Alternatively,transgenic animals are produced wherein a polynucleotide encoding thedesired specific binding agent is introduced into the genome of arecipient animal, such as, for example, a mouse, rabbit, goat, or cow,in a manner that permits expression of the polynucleotide moleculesencoding a monoclonal antibody or other specific binding agent. In oneaspect, the polynucleotides encoding the monoclonal antibody or otherspecific binding agent can be ligated to mammary-specific regulatorysequences, and the chimeric polynucleotides can be introduced into thegermline of the target animal. The resulting transgenic animal thenproduces the desired antibody in its milk [Pollock et al., J ImmunolMeth 231:147-157 (1999); Little et al., Immunol Today 8:364-370 (2000)].In addition, plants may be used to express and produce Ang-2 specificbinding agents such as monoclonal antibodies by transfecting suitableplants with the polynucleotides encoding the monoclonal antibodies orother specific binding agents.

In another embodiment of the present invention, a monoclonal orpolyclonal antibody or fragment thereof that is derived from other thana human species may be “humanized” or “chimerized”. Methods forhumanizing non-human antibodies are well known in the art. (see U.S.Pat. Nos. 5,859,205, 5,585,089, and 5,693,762). Humanization isperformed, for example, using methods described in the art [Jones etal., Nature 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327(1988); Verhoeyen et al., Science 239:1534-1536 (1988)] by substitutingat least a portion of, for example a rodent, complementarity-determiningregion (CDRs) for the corresponding regions of a human antibody. Theinvention also provides variants and derivatives of these humanantibodies as discussed herein and well known in the art.

Also encompassed by the invention are fully human antibodies that bindAng-2 polypeptides, as well as, antigen-binding fragments, variantsand/or derivatives thereof. Such antibodies can be produced using thephage display technique described above. Alternatively, transgenicanimals (e.g., mice) that are capable of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production can beused to generate such antibodies. This can be accomplished byimmunization of the animal with an Ang-2 antigen or fragments thereofwhere the Ang-2 fragments have an amino acid sequence that is unique toAng-2. Such immunogens can be optionally conjugated to a carrier. See,for example, Jakobovits et al., Proc Natl Acad Sci (USA), 90: 2551-2555(1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggermann etal., Year in Immuno, 7: 33 (1993). In one method, such transgenicanimals are produced by incapacitating the endogenous loci encoding theheavy and light immunoglobulin chains therein, and inserting lociencoding human heavy and light chain proteins into the genome thereof.Partially modified animals, that are those having less than the fullcomplement of these modifications, are then crossbred to obtain ananimal having all of the desired immune system modifications. Whenadministered an immunogen, these transgenic animals are capable ofproducing antibodies with human variable regions, including human(rather than e.g., murine) amino acid sequences, that areimmuno-specific for the desired antigens. See PCT application Nos.,PCT/US96/05928 and PCT/US93/06926. Additional methods are described inU.S. Pat. No. 5,545,807, PCT application Nos. PCT/US91/245,PCT/GB89/01207, and in EP 546073B1 and EP 546073A1. Human antibodies mayalso be produced by the expression of recombinant DNA in host cells orby expression in hybridoma cells as described herein.

Transgenesis is achieved in a number of different ways. See, forexample, Bruggeman et al., Immunol Today 17:391-7 (1996). In oneapproach, a minilocus is constructed such that gene segments in agermline configuration are brought artificially close to each other. Dueto size limitations (i.e., having generally less than 30 kb), theresulting minilocus will contain a limited number of differing genesegments, but is still capable of producing a large repertoire ofantibodies. Miniloci containing only human DNA sequences, includingpromoters and enhancers are fully functional in the transgenic mouse.

When larger number of gene segments are desired in the transgenicanimal, yeast artificial chromosomes (YACs) are utilized. YACs can rangefrom several hundred kilobases to 1 Mb and are introduced into the mouse(or other appropriate animal) genome via microinjection directly into anegg or via transfer of the YAC into embryonic stem (ES)-cell lines. Ingeneral, YACs are transferred into ES cells by lipofection of thepurified DNA, or yeast spheroplast fusion wherein the purified DNA iscarried in micelles and fusion is carried out in manner similar tohybridoma fusion protocols. Selection of desired ES cells following DNAtransfer is accomplished by including on the YAC any of the selectablemarkers known in the art.

As another alternative, bacteriophage P1 vectors are used which areamplified in a bacterial E. coli host. While these vectors generallycarry less inserted DNA than a YAC, the clones are readily grown in highenough yield to permit direct microinjection into a mouse egg. Use of acocktail of different P1 vectors has been shown to lead to high levelsof homologous recombination.

Once an appropriate transgenic mouse (or other appropriate animal) hasbeen identified, using any of the techniques known in the art to detectserum levels of a circulating antibody (e.g., ELISA), the transgenicanimal is crossed with a mouse in which the endogenous Ig locus has beendisrupted. The result provides progeny wherein essentially all B cellsexpress human antibodies.

As still another alternative, the entire animal Ig locus is replacedwith the human Ig locus, wherein the resulting animal expresses onlyhuman antibodies. In another approach, portions of the animal's locusare replaced with specific and corresponding regions in the human locus.In certain cases, the animals resulting from this procedure may expresschimeric antibodies, as opposed to fully human antibodies, depending onthe nature of the replacement in the mouse Ig locus.

Human antibodies can also be produced by exposing human splenocytes (Bor T cells) to an antigen in vitro, then reconstituting the exposedcells in an immunocompromised mouse, e.g. SCID or nod/SCID. See Brams etal., J Immunol, 160: 2051-2058 [1998]; Carballido et al., Nat Med, 6:103-106 [2000]. In one approach, engraftment of human fetal tissue intoSCID mice (SCID-hu) results in long-term hematopoiesis and human T-celldevelopment [McCune et al., Science 241:1532-1639 (1988); Ifversen etal., Sem Immunol 8:243-248 (1996)]. Any humoral immune response in thesechimeric mice is completely dependent on co-development of T-cells inthe animals [Martensson et al., Immunol 83:1271-179 (1994)]. In analternative approach, human peripheral blood lymphocytes aretransplanted intraperitoneally (or otherwise) into SCID mice [Mosier etal., Nature 335:256-259 (1988)]. When the transplanted cells are treatedwith either a priming agent, such as Staphylococcal Enterotoxin A (SEA)[Martensson et al., Immunol 84: 224-230 (1995)], or anti-human CD40monoclonal antibodies [Murphy et al., Blood 86:1946-1953 (1995)], higherlevels of B cell production are detected.

Alternatively, an entirely synthetic human heavy chain repertoire iscreated from unrearranged V gene segments by assembling each human VHsegment with D segments of random nucleotides together with a human Jsegment [Hoogenboom et al., J Mol Biol 227:381-388 (1992)]. Likewise, alight chain repertoire is constructed by combining each human V segmentwith a J segment [Griffiths et al., EMBO J 13:3245-3260 (1994)].Nucleotides encoding the complete antibody (i.e., both heavy and lightchains) are linked as a single chain Fv fragment and this polynucleotideis ligated to a nucleotide encoding a filamentous phage minor coatprotein. When this fusion protein is expressed on the surface of thephage, a polynucleotide encoding a specific antibody is identified byselection using an immobilized antigen.

In still another approach, antibody fragments are assembled as two Fabfragments by fusion of one chain to a phage protein and secretion of theother into bacterial periplasm [Hoogenboom et al., Nucl Acids Res19:4133-4137 [1991]; Barbas et al., Proc Natl Acad Sci (USA)88:7978-7982 (1991)].

Large-scale production of chimeric, humanized, CDR-grafted, and fullyhuman antibodies, or antigen-binding fragments thereof, are typicallyproduced by recombinant methods. Polynucleotide molecule(s) encoding theheavy and light chains of each antibody or antigen-binding fragmentsthereof, can be introduced into host cells and expressed using materialsand procedures described herein. In a preferred embodiment, theantibodies are produced in mammalian host cells, such as CHO cells.Details of such production are described herein.

The specific binding agents of the present invention, such as theantibodies, antibody fragments, and antibody derivatives of theinvention can further comprise any constant region known in the art. Thelight chain constant region can be, for example, a kappa- or lambda-typelight chain constant region, e.g., a human kappa- or lambda-type lightchain constant region. The heavy chain constant region can be, forexample, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chainconstant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, ormu-type heavy chain constant region. In one embodiment, the light orheavy chain constant region is a fragment, derivative, variant, ormutein of a naturally occurring constant region.

In one embodiment, the specific binding agents of the present invention,such as the antibodies, antibody fragments, and antibody derivatives ofthe invention comprise an IgG.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.

The specific binding agents of the present invention, such as theantibodies, antibody fragments, and antibody derivatives of theinvention may comprise the IgG1 heavy chain constant domain or afragment of the IgG1 heavy chain domain. The antibodies, antibodyfragments, and antibody derivatives of the invention may furthercomprise the constant light chain kappa or lambda domains or a fragmentof these. Light chain constant regions and polynucleotides encoding themare provided herein below. In another embodiment, the antibodies,antibody fragments, and antibody derivatives of the invention furthercomprise a heavy chain constant domain, or a fragment thereof, such asthe IgG2 heavy chain constant region also shown herein below.

The nucleic acid (DNA) encoding constant heavy and constant light chaindomains, and the amino acids sequences of heavy and light chain domainsare provided herein below. Lambda variable domains can be fused tolambda constant domains and kappa variable domains can be fused to kappaconstant domains.

IgG2 Heavy Constant domain DNA (SEQ ID NO: 41):gctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagegcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaIgG2 Heavy Constant domain Protein (SEQ ID NO: 42):ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKappa Light Constant domain DNA (SEQ ID NO: 43):cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagKappa Light Constant domain Protein (SEQ ID NO: 44):RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLambda Light Constant domain DNA (SEQ ID NO: 45):ggccaaccgaaagcggcgccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatagLambda Light Constant domain Protein (SEQ ID NO: 46):GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

The specific binding agents of the present invention, such as theantibodies, antibody fragments, and antibody derivatives of theinvention include those comprising, for example, the variable domaincombinations H6L7, H5L7, H4L13, H11L7, H4L7, H10L7, H5L6, H2L7, H5L8,H6L8, H3L7, H5L4, H4L12, H6L6, H4L2, H4L6, H4L4, H5L11, H5L1, H4L11,H5L12, H5L9 having a desired isotype (for example, IgA, IgG1, IgG2,IgG3, IgG4, IgM, IgE, and IgD) as well as Fab or F(ab′)₂ fragmentsthereof. Moreover, if an IgG4 is desired, it may also be desired tointroduce a point mutation in the hinge region as described in Bloom etal., 1997, Protein Science 6:407 (incorporated by reference herein) toalleviate a tendency to form intra-H chain disulfide bonds that can leadto heterogeneity in the IgG4 antibodies.

Additional Useful Sequence Information

The following sequences of the IgG1, IgG2, IgG3, and IgG4 isotypes areused in combination with the variable heavy chain sequences of theantibodies of the present invention to make a specific desired isotypeof said antibody:

Human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Human IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

HC Sequences of the Antibodies of the Present Invention as an IgG2

The following sequences represent the heavy chain sequences of theantibodies of the present invention as IgG2 isotype. The light chainsequences remain the same, which are provided in the Examples. Theunderlined sequence portions represent the IgG2 sequences:

H2 EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIEYADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H3EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIQYADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H6EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDIYTGYGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H10EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H11EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGMWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H4EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDLLTGYGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H5EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDIWTGYGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Fusion Partners of Specific Binding Agents

In a further embodiment of the invention, the polypeptides comprisingthe amino acid sequence variable domains of Ang-2 antibodies, such as aheavy chain variable region with an amino acid sequence as describedherein or a light chain variable region with an amino acid sequence asdescribed herein, may be fused at either the N-terminus or theC-terminus to one or more domains of an Fc region of human IgG. Whenconstructed together with a therapeutic protein such as the Fab of anAng-2-specific antibody, an Fc domain can provide longer half-life orincorporate such functions as Fc receptor binding, Protein A binding,complement fixation and perhaps even placental transfer. [Capon et al.,Nature, 337: 525-531 (1989)].

In one example, the antibody hinge, CH2 and CH3 regions may be fused ateither the N-terminus or C-terminus of the specific binding agentpolypeptides such as an anti-Ang-2 Fab or Fv fragment (obtained, e.g.,from a phage display library) using methods known to the skilledartisan. The resulting fusion protein may be purified by use of aProtein A or Protein G affinity column. Peptides and proteins fused toan Fc region have been found to exhibit a substantially greaterhalf-life in vivo than the unfused counterpart. Also, a fusion to an Fcregion allows for dimerization/multimerization of the fusionpolypeptide. The Fc region may be a naturally occurring Fc region, ormay be altered to improve certain qualities, such as therapeuticqualities, circulation time, decrease aggregation problems, etc. Otherexamples known in the art include those wherein the Fc region, which maybe human or another species, or may be synthetic, is fused to theN-terminus of CD30L to treat Hodgkin's Disease, anaplastic lymphoma andT-cell leukemia (U.S. Pat. No. 5,480,981), the Fc region is fused to theTNF receptor to treat septic shock [Fisher et al., N Engl J Med, 334:1697-1702 (1996)], and the Fc region is fused to the Cd4 receptor totreat AIDS [Capon et al., Nature, 337: 525-31 (1989)].

Catalytic antibodies are another type of fusion molecule and includeantibodies to which one or more cytotoxic, or more generally one or morebiologically active, moieties are attached to the specific bindingagent. See, for example Rader et al., Chem Eur J 12:2091-2095 (2000).Cytotoxic agents of this type improve antibody-mediated cytotoxicity,and include such moieties as cytokines that directly or indirectlystimulate cell death, radioisotopes, chemotherapeutic drugs (includingprodrugs), bacterial toxins (ex. pseudomonas exotoxin, diphtheria toxin,etc.), plant toxins (ex. ricin, gelonin, etc.), chemical conjugates(e.g., maytansinoid toxins, calechaemicin, etc.), radioconjugates,enzyme conjugates (RNase conjugates, antibody-directed enzyme/prodrugtherapy [ADEPT)]), and the like. In one aspect, the cytotoxic agent canbe “attached” to one component of a bi-specific or multi-specificantibody by binding of this agent to one of the alternative antigenrecognition sites on the antibody. As an alternative, protein cytotoxinscan be expressed as fusion proteins with the specific binding agentfollowing ligation of a polynucleotide encoding the toxin to apolynucleotide encoding the binding agent. In still another alternative,the specific binding agent can be covalently modified to include thedesired cytotoxin.

Examples of such fusion proteins are immunogenic polypeptides, proteinswith long circulating half lives, such as immunoglobulin constantregions, marker proteins, proteins or polypeptides that facilitatepurification of the desired specific binding agent polypeptide, andpolypeptide sequences that promote formation of multimeric proteins(such as leucine zipper motifs that are useful in dimerformation/stability).

This type of insertional variant generally has all or a substantialportion of the native molecule, linked at the N- or C-terminus, to allor a portion of a second polypeptide. For example, fusion proteinstypically employ leader sequences from other species to permit therecombinant expression of a protein in a heterologous host. Anotheruseful fusion protein includes the addition of an immunologically activedomain, such as an antibody epitope, to facilitate purification of thefusion protein. Inclusion of a cleavage site at or near the fusionjunction will facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals or transmembrane regions.

There are various commercially available fusion protein expressionsystems that may be used in the present invention. Particularly usefulsystems include but are not limited to the glutathione-S-transferase(GST) system (Pharmacia), the maltose binding protein system (NEB,Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×Hissystem (Qiagen, Chatsworth, Calif.). These systems are capable ofproducing recombinant polypeptides bearing only a small number ofadditional amino acids, which are unlikely to affect the antigenicability of the recombinant polypeptide. For example, both the FLAGsystem and the 6×His system add only short sequences, both of which areknown to be poorly antigenic and which do not adversely affect foldingof the polypeptide to its native conformation. Another N-terminal fusionthat is contemplated to be useful is the fusion of a Met-Lys dipeptideat the N-terminal region of the protein or peptides. Such a fusion mayproduce beneficial increases in protein expression or activity.

A particularly useful fusion construct may be one in which a specificbinding agent peptide is fused to a hapten to enhance immunogenicity ofa specific binding agent fusion construct which is useful, for example,in the production of anti-idiotype antibodies of the invention. Suchfusion constructs to increase immunogenicity are well known to those ofskill in the art, for example, a fusion of specific binding agent with ahelper antigen such as hsp70 or peptide sequences such as fromdiphtheria toxin chain or a cytokine such as IL-2 will be useful ineliciting an immune response. In other embodiments, fusion construct canbe made which will enhance the targeting of the antigen binding agentcompositions to a specific site or cell.

Other fusion constructs including heterologous polypeptides with desiredproperties, e.g., an Ig constant region to prolong serum half-life or anantibody or fragment thereof for targeting also are contemplated. Otherfusion systems produce polypeptide hybrids where it is desirable toexcise the fusion partner from the desired polypeptide. In oneembodiment, the fusion partner is linked to the recombinant specificbinding agent polypeptide by a peptide sequence containing a specificrecognition sequence for a protease. Examples of suitable sequences arethose recognized by the Tobacco Etch Virus protease (Life Technologies,Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

The invention also provides fusion polypeptides comprising all or partof a variable domain of an Ang-2 antibody, such as a heavy chainvariable region with an amino acid sequence as described herein or alight chain variable region with an amino acid sequence as describedherein in combination with truncated tissue factor (tTF), a vasculartargeting agent consisting of a truncated form of a humancoagulation-inducing protein that acts as a tumor blood vessel clottingagent. The fusion of tTF to the anti-Ang-2 antibody, or fragmentsthereof may facilitate the delivery of anti-Ang-2 to target cells.

Variants of Specific Binding Agents

Variants of Specific Binding Agents of the present invention includeinsertion, deletion, and/or substitution variants. In one aspect of theinvention, insertion variants are provided wherein one or more aminoacid residues supplement a specific binding agent amino acid sequence.Insertions may be located at either or both termini of the protein, ormay be positioned within internal regions of the specific binding agentamino acid sequence. Insertional variants with additional residues ateither or both termini can include, for example, fusion proteins andproteins including amino acid tags or labels. Insertion variants includespecific binding agent polypeptides wherein one or more amino acidresidues are added to a specific binding agent amino acid sequence, orfragment thereof.

Variant products of the invention also include mature specific bindingagent products. Such specific binding agent products have the leader orsignal sequences removed, however the resulting protein has additionalamino terminal residues as compared to wild-type Ang-2 polypeptide. Theadditional amino terminal residues may be derived from another protein,or may include one or more residues that are not identifiable as beingderived from a specific protein. Specific binding agent products with anadditional methionine residue at position −1 (Mer⁻¹-specific bindingagent) are contemplated, as are specific binding agent products withadditional methionine and lysine residues at positions −2 and −1 (Met2-Lys⁻¹-specific binding agent). Variants of specific binding agentshaving additional Met, Met-Lys, Lys residues (or one or more basicresidues in general) are particularly useful for enhanced recombinantprotein production in bacterial host cells.

The invention also embraces specific binding agent variants havingadditional amino acid residues that arise from use of specificexpression systems. For example, use of commercially available vectorsthat express a desired polypeptide as part of glutathione-S-transferase(GST) fusion product provides the desired polypeptide having anadditional glycine residue at amino acid position −1 after cleavage ofthe GST component from the desired polypeptide. Variants which resultfrom expression in other vector systems are also contemplated, includingthose wherein poly-histidine tags are incorporated into the amino acidsequence, generally at the carboxy and/or amino terminus of thesequence.

Insertional variants also include fusion proteins as described above,wherein the amino and/or carboxy termini of the specific bindingagent-polypeptide is fused to another polypeptide, a fragment thereof,or amino acid sequences which are not generally recognized to be part ofany specific protein sequence.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a specific binding agent polypeptide areremoved. Deletions can be effected at one or both termini of thespecific binding agent polypeptide, or from removal of one or moreresidues within the specific binding agent amino acid sequence. Deletionvariants necessarily include all fragments of a specific binding agentpolypeptide.

Antibody fragments include those portions of the antibody that bind toan epitope on the antigen polypeptide. Examples of such fragmentsinclude Fab and F(ab′)₂ fragments generated, for example, by enzymaticor chemical cleavage of full-length antibodies. Other binding fragmentsinclude those generated by recombinant DNA techniques, such as theexpression of recombinant plasmids containing nucleic acid sequencesencoding antibody variable regions. The invention also embracespolypeptide fragments of an Ang-2 binding agent wherein the fragmentsmaintain the ability to specifically bind an Ang-2 polypeptide.Fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 ormore consecutive amino acids of a peptide or polypeptide of theinvention are comprehended herein. Preferred polypeptide fragmentsdisplay immunological properties unique to or specific for theantigen-binding agent so of the invention. Fragments of the inventionhaving the desired immunological properties can be prepared by any ofthe methods well known and routinely practiced in the art.

In still another aspect, the invention provides substitution variants ofspecific binding agents of the invention. Substitution variants aregenerally considered to be “similar” to the original polypeptide or tohave a certain “percent identity” to the original polypeptide, andinclude those polypeptides wherein one or more amino acid residues of apolypeptide are removed and replaced with alternative residues. In oneaspect, the substitutions are conservative in nature, however, theinvention embraces substitutions that are also non-conservative.

Identity and similarity of related polypeptides can be readilycalculated by known methods. Such methods include, but are not limitedto, those described in Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York (1988); Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York (1993);Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press (1987); SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press,New York (1991); and Carillo et al., SIAM J. Applied Math., 48:1073(1988).

Preferred methods to determine the relatedness or percent identity oftwo polypeptides are designed to give the largest match between thesequences tested. Methods to determine identity are described inpublicly available computer programs. Preferred computer program methodsto determine identity between two sequences include, but are not limitedto, the GCG program package, including GAP (Devereux et al., Nucl. Acid.Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin,Madison, Wis., BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol.Biol., 215:403-410 (1990)). The BLASTX program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;Altschul et al., supra (1990)). The well-known Smith Waterman algorithmmay also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least tenpercent of the full length of the target polypeptide being compared,i.e., at least 40 contiguous amino acids where sequences of at least 400amino acids are being compared, 30 contiguous amino acids wheresequences of at least 300 to about 400 amino acids are being compared,at least 20 contiguous amino acids where sequences of 200 to about 300amino acids are being compared, and at least 10 contiguous amino acidswhere sequences of about 100 to 200 amino acids are being compared.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is typically calculated as 3× the average diagonal; the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually 1/10 times the gap openingpenalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62are used in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see Dayhoff et al., Atlas of ProteinSequence and Structure, 5(3)(1978) for the PAM 250 comparison matrix;Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919 (1992) forthe BLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program may be useful with the above parameters. In certainembodiments, the aforementioned parameters are the default parametersfor polypeptide comparisons (along with no penalty for end gaps) usingthe GAP algorithm.

In certain embodiments, the parameters for polynucleotide moleculesequence comparisons include the following:

-   -   Algorithm: Needleman et al., supra (1970);    -   Comparison matrix: matches=+10, mismatch=0    -   Gap Penalty: 50    -   Gap Length Penalty: 3

The GAP program may also be useful with the above parameters. Theaforementioned parameters are the default parameters for polynucleotidemolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference forany purpose.

The amino acids may have either L or D stereochemistry (except for Gly,which is neither L nor D) and the polypeptides and compositions of thepresent invention may comprise a combination of stereochemistries.However, the L stereochemistry is preferred. The invention also providesreverse molecules wherein the amino terminal to carboxy terminalsequence of the amino acids is reversed. For example, the reverse of amolecule having the normal sequence X₁-X₂-X₃ would be X₃-X₂-X₁. Theinvention also provides retro-reverse molecules wherein, as above, theamino terminal to carboxy terminal sequence of amino acids is reversedand residues that are normally “L” enantiomers are altered to the “D”stereoisomer form.

Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include, withoutlimitation: aminoadipic acid, beta-alanine, beta-aminopropionic acid,aminobutyric acid, piperidinic acid, aminocaprioic acid, aminoheptanoicacid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid,desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine,N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline,isodesmosine, allo-isoleucine, N-methylglycine, sarcosine,N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine,4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and amino acids (e.g., 4-hydroxyproline).

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the humanantibody that are homologous with non-human antibodies, or into thenon-homologous regions of the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln, Glu, Asp Gln Asp Glu, Gln, Asn Glu Cys Ser, Ala Ser Gln Asn,Glu, Asp Asn Glu Asp, Asn, Gln Asp Gly Pro, Ala Ala His Asn, Gln, Lys,Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile,Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, AsnMet Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly SerThr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, SerPhe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In certain embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues which are important for activity or structure insimilar proteins. One skilled in the art may opt for chemically similaramino acid substitutions for such predicted important amino acidresidues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. In certain embodiments, one skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change may be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and “evolutionary linkage” (See Holm, supra (1999), andBrenner, supra (1997)).

In certain embodiments, antibody variants include glycosylation variantswherein the number and/or type of glycosylation site has been alteredcompared to the amino acid sequences of the parent polypeptide. Incertain embodiments, protein variants comprise a greater or a lessernumber of N-linked glycosylation sites than the native protein. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionswhich eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred antibodyvariants include cysteine variants wherein one or more cysteine residuesare deleted from or substituted for another amino acid (e.g., serine) ascompared to the parent amino acid sequence. Cysteine variants may beuseful when antibodies must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

According to certain embodiments, amino acid substitutions are thosewhich: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (5) confer ormodify other functional properties on such polypeptides. According tocertain embodiments, single or multiple amino acid substitutions (incertain embodiments, conservative amino acid substitutions) may be madein the naturally-occurring sequence (in certain embodiments, in theportion of the polypeptide outside the domain(s) forming intermolecularcontacts). In certain embodiments, a conservative amino acidsubstitution typically may not substantially change the structuralcharacteristics of the parent sequence (e.g., a replacement amino acidshould not tend to break a helix that occurs in the parent sequence, ordisrupt other types of secondary structure that characterizes the parentsequence). Examples of art-recognized polypeptide secondary and tertiarystructures are described in Proteins, Structures and MolecularPrinciples (Creighton, Ed., W.H. Freeman and Company, New York (1984));Introduction to Protein Structure (C. Branden and J. Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature354:105 (1991).

The specific binding agent molecules of this invention that arepolypeptide or peptide substitution variants may have up to about ten totwelve percent of the original amino acid sequence replaced. Forantibody variants, the heavy chain may have 50, 49, 48, 47, 46, 45, 44,43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26,25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 amino acid replaced, while the light chain may have25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 amino acid replaced.

Derivatives of Specific Binding Agents

The invention also provides derivatives of specific binding agentpolypeptides. Derivatives include specific binding agent polypeptidesbearing modifications other than insertion, deletion, or substitution ofamino acid residues. Preferably, the modifications are covalent innature, and include for example, chemical bonding with polymers, lipids,other organic, and inorganic moieties. Derivatives of the invention maybe prepared to increase circulating half-life of a specific bindingagent polypeptide, or may be designed to improve targeting capacity forthe polypeptide to desired cells, tissues, or organs.

The invention further embraces derivative binding agents covalentlymodified to include one or more water soluble polymer attachments suchas polyethylene glycol, polyoxyethylene glycol, or polypropylene glycolas described U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417,4,791,192 and 4,179,337. Still other useful polymers known in the artinclude monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol, as well as mixtures of these polymers. Particularlypreferred are specific binding agent products covalently modified withpolyethylene glycol (PEG) subunits. Water-soluble polymers may be bondedat specific positions, for example at the amino terminus of the specificbinding agent products, or randomly attached to one or more side chainsof the polypeptide. The use of PEG for improving the therapeuticcapacity for specific binding agent, and for humanized antibodies inparticular, is described in U.S. Pat. No. 6,133,426 to Gonzales et al.,issued Oct. 17, 2000.

Target Sites for Antibody Mutagenesis

Certain strategies can be employed to manipulate inherent properties ofan Ang-1 and/or Ang-2-specific antibody, such as the affinity of theantibody for its target. These strategies include the use ofsite-specific or random mutagenesis of the polynucleotide moleculeencoding the antibody to generate antibody variants, followed by ascreening step designed to recover antibody variants that exhibit thedesired change, e.g. increased or decreased affinity.

The amino acid residues most commonly targeted in mutagenic strategiesare those in the CDRs. As described supra, these regions contain theresidues that actually interact with Ang-1 and/or Ang-2 and other aminoacids that affect the spatial arrangement of these residues. However,amino acids in the framework regions of the variable domains outside theCDR regions have also been shown to make substantial contributions tothe antigen-binding properties of the antibody, and can be targeted tomanipulate such properties. See Hudson, Curr Opin Biotech, 9:395-402(1999) and references therein.

Smaller and more effectively screened libraries of antibody variants canbe produced by restricting random or site-directed mutagenesis to sitesin the CDRs that correspond to areas prone to “hyper-mutation” duringthe somatic affinity maturation process. See Chowdhury and Pastan,Nature Biotech, 17: 568-572 [1999] and references therein. The types ofDNA elements known to define hyper-mutation sites in this manner includedirect and inverted repeats, certain consensus sequences, secondarystructures, and palindromes. The consensus DNA sequences include thetetrabase sequence Purine-G-Pyrimidine-A/T (i.e. A or G-G-C or T-A or T)and the serine codon AGY (wherein Y can be a C or a T).

Thus, an embodiment of the present invention includes mutagenicstrategies with the goal of increasing the affinity of an antibody forits target. These strategies include mutagenesis of the entire variableheavy and light chain, mutagenesis of the CDR regions only, mutagenesisof the consensus hypermutation sites within the CDRs, mutagenesis offramework regions, or any combination of these approaches (“mutagenesis”in this context could be random or site-directed). Definitivedelineation of the CDR regions and identification of residues comprisingthe binding site of an antibody can be accomplished though solving thestructure of the antibody in question, and the antibody-ligand complex,through techniques known to those skilled in the art, such as X-raycrystallography. Various methods based on analysis and characterizationof such antibody crystal structures are known to those of skill in theart and can be employed, although not definitive, to approximate the CDRregions. Examples of such commonly used methods include the Kabat,Chothia, AbM and contact definitions.

The Kabat definition is based on the sequence variability and is themost commonly used definition to predict CDR regions. [Johnson and Wu,Nucleic Acids Res, 28: 214-8 (2000)]. The Chothia definition is based onthe location of the structural loop regions. [Chothia et al., J MolBiol, 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989)].The AbM definition is a compromise between the Kabat and Chothiadefinition. AbM is an integral suite of programs for antibody structuremodeling produced by Oxford Molecular Group [Martin et al., Proc NatlAcad Sci (USA) 86:9268-9272 (1989); Rees, et al., ABM™, a computerprogram for modeling variable regions of antibodies, Oxford, UK; OxfordMolecular, Ltd.]. The AbM suite models the tertiary structure of anantibody from primary sequencing using a combination of knowledgedatabases and ab initio methods. An additional definition, known as thecontact definition, has been recently introduced. [MacCallum et al., JMol Biol, 5:732-45 (1996)]. This definition is based on an analysis ofthe available complex crystal structures.

By convention, the CDR regions in the heavy chain are typically referredto as H1, H2 and H3 and are numbered sequentially in order counting fromthe amino terminus to the carboxy terminus. The CDR regions in the lightchain are typically referred to as L1, L2 and L3 and are numberedsequentially in order counting from the amino terminus to the carboxyterminus.

The CDR-H1 is approximately 10 to 12 residues in length and typicallystarts 4 residues after a Cys according to the Chothia and AbMdefinitions or typically 5 residues later according to the Kabatdefinition. The H1 is typically followed by a Trp, typically Trp-Val,but also Trp-Ile, or Trp-Ala. The length of H1 is approximately 10 to 12residues according to the AbM definition while the Chothia definitionexcludes the last 4 residues.

The CDR-H2 typically starts 15 residues after the end of H1 according tothe Kabat and AbM definition. The residues preceding H2 are typicallyLeu-Glu-Trp-Ile-Gly but there are a number of variations. H2 istypically followed by the amino acid sequenceLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. According to the Kabatdefinition, the length of the H2 is approximately 16 to 19 residueswhere the AbM definition predicts the length to be typically 9 to 12residues.

The CDR-H3 typically starts 33 residues after the end of H2 and istypically preceded by the amino acid sequence (typically Cys-Ala-Arg).The H3 is typically followed by the amino acid sequence-Gly. The lengthof H3 can be anywhere between 3 to 25 residues.

The CDR-L1 typically starts at approximately residue 24 and willtypically follow a Cys. The residue after the CDR-L1 is always a Trp andwill typically begin the sequence Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln,or Trp-Tyr-Leu. The length of CDR-L1 is approximately 10 to 17 residues.The punitive CDR-L1 for the antibodies of the invention follows thispattern exactly with a Cys residue followed by 15 amino acids thenTrp-Tyr-Gln.

The CDR-L2 starts approximately 16 residues after the end of L1. It willgenerally follow residues Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe. Thelength of CDR-L2 is approximately 7 residues.

The CDR-L3 typically starts 33 residues after the end of L2 andtypically follows a Cys. L3 is typically followed by the amino acidsequence Phe-Gly-XXX-Gly. The length of L3 is approximately 7 to 11residues.

Various methods for modifying antibodies have been described in the art.For example, U.S. Pat. No. 5,530,101 (to Queen et al., Jun. 25, 1996)describes methods to produce humanized antibodies wherein the sequenceof the humanized immunoglobulin heavy chain variable region framework is65% to 95% identical to the sequence of the donor immunoglobulin heavychain variable region framework. Each humanized immunoglobulin chainwill usually comprise, in addition to the CDRs, amino acids from thedonor immunoglobulin framework that are, e.g., capable of interactingwith the CDRs to affect binding affinity, such as one or more aminoacids which are immediately adjacent to a CDR in the donorimmunoglobulin or those within about 3 angstroms as predicted bymolecular modeling. The heavy and light chains may each be designed byusing any one or all of various position criteria. When combined into anintact antibody, the humanized immunoglobulins of the present inventionwill be substantially non-immunogenic in humans and retain substantiallythe same affinity as the donor immunoglobulin to the antigen, such as aprotein or other compound containing an epitope. See also, relatedmethods in U.S. Pat. No. 5,693,761 to Queen, et al., issued Dec. 2, 1997(“Polynucleotides encoding improved humanized immunoglobulins”); U.S.Pat. No. 5,693,762 to Queen, et al., issued Dec. 2, 1997 (“HumanizedImmunoglobulins”); U.S. Pat. No. 5,585,089 to Queen, et al. issued Dec.17, 1996 (“Humanized Immunoglobulins”).

In one example, U.S. Pat. No. 5,565,332 to Hoogenboom et al. issued Oct.15, 1996 (“Production of chimeric antibodies—a combinatorial approach”)describes methods for the production of antibodies, and antibodyfragments which have similar binding specificity as a parent antibodybut which have increased human characteristics. Humanized antibodies areobtained by chain shuffling, using, for example, phage displaytechnology, and a polypeptide comprising a heavy or light chain variabledomain of a non-human antibody specific for an antigen of interest iscombined with a repertoire of human complementary (light or heavy) chainvariable domains. Hybrid pairings that are specific for the antigen ofinterest are identified and human chains from the selected pairings arecombined with a repertoire of human complementary variable domains(heavy or light). In another embodiment, a component of a CDR from anon-human antibody is combined with a repertoire of component parts ofCDRs from human antibodies. From the resulting library of antibodypolypeptide dimers, hybrids are selected and used in a second humanizingshuffling step. Alternatively, this second step is eliminated if thehybrid is already of sufficient human character to be of therapeuticvalue. Methods of modification to increase human character are alsodescribed. See also Winter, FEBS Letts 430:92-92 (1998).

As another example, U.S. Pat. No. 6,054,297 to Carter et al., issuedApr. 25, 2000 describes a method for making humanized antibodies bysubstituting a CDR amino acid sequence for the corresponding human CDRamino acid sequence and/or substituting a FR amino acid sequence for thecorresponding human FR amino acid sequences.

As another example, U.S. Pat. No. 5,766,886 to Studnicka et al., issuedJun. 16, 1998 (“Modified antibody variable domains”) describes methodsfor identifying the amino acid residues of an antibody variable domainwhich may be modified without diminishing the native affinity of theantigen binding domain while reducing its immunogenicity with respect toa heterologous species and methods for preparing these modified antibodyvariable domains which are useful for administration to heterologousspecies. See also U.S. Pat. No. 5,869,619 to Studnicka issued Feb. 9,1999.

As discussed, modification of an antibody by any of the methods known inthe art is typically designed to achieve increased binding affinity foran antigen and/or reduce immunogenicity of the antibody in therecipient. In one approach, humanized antibodies can be modified toeliminate glycosylation sites in order to increase affinity of theantibody for its cognate antigen [Co et al., Mol Immunol 30:1361-1367(1993)]. Techniques such as “reshaping,” “hyperchimerization,” and“veneering/resurfacing” have produced humanized antibodies with greatertherapeutic potential. [Vaswami et al., Annals of Allergy, Asthma, &Immunol 81:105 (1998); Roguska et al., Prot Engineer 9:895-904 (1996)].See also U.S. Pat. No. 6,072,035 to Hardman et al., issued Jun. 6, 2000,which describes methods for reshaping antibodies. While these techniquesdiminish antibody immunogenicity by reducing the number of foreignresidues, they do not prevent anti-idiotypic and anti-allotypicresponses following repeated administration of the antibodies.Alternatives to these methods for reducing immunogenicity are describedin Gilliland et al., J Immunol 62(6): 3663-71 (1999).

In many instances, humanizing antibodies result in a loss of antigenbinding capacity. It is therefore preferable to “back mutate” thehumanized antibody to include one or more of the amino acid residuesfound in the original (most often rodent) antibody in an attempt torestore binding affinity of the antibody. See, for example, Saldanha etal., Mol Immunol 36:709-19 (1999).

Non-Peptide Specific Binding Agent Analogs/Protein Mimetics

Furthermore, nonpeptide specific binding agent analogs of peptides thatprovide a stabilized structure or lessened bio-degradation, are alsocontemplated. Specific binding agent peptide mimetic analogs can beprepared based on a selected inhibitory peptide by replacement of one ormore residues by nonpeptide moieties. Preferably, the nonpeptidemoieties permit the peptide to retain its natural confirmation, orstabilize a preferred, e.g., bioactive, confirmation which retains theability to recognize and bind Ang-1 and/or Ang-2. In one aspect, theresulting analog/mimetic exhibits increased binding affinity for Ang-1and/or Ang-2. One example of methods for preparation of nonpeptidemimetic analogs from specific binding agent peptides is described inNachman et al., Regul Pept 57:359-370 (1995). If desired, the specificbinding agent peptides of the invention can be modified, for instance,by glycosylation, amidation, carboxylation, or phosphorylation, or bythe creation of acid addition salts, amides, esters, in particularC-terminal esters, and N-acyl derivatives of the peptides of theinvention. The specific binding agent peptides also can be modified tocreate peptide derivatives by forming covalent or noncovalent complexeswith other moieties. Covalently-bound complexes can be prepared bylinking the chemical moieties to functional groups on the side chains ofamino acids comprising the specific binding agent peptides, or at the N-or C-terminus

In particular, it is anticipated that the specific binding agentpeptides can be conjugated to a reporter group, including, but notlimited to a radiolabel, a fluorescent label, an enzyme (e.g., thatcatalyzes a colorimetric or fluorometric reaction), a substrate, a solidmatrix, or a carrier (e.g., biotin or avidin). The invention accordinglyprovides a molecule comprising an antibody molecule, wherein themolecule preferably further comprises a reporter group selected from thegroup consisting of a radiolabel, a fluorescent label, an enzyme, asubstrate, a solid matrix, and a carrier. Such labels are well known tothose of skill in the art, e.g., biotin labels are particularlycontemplated. The use of such labels is well known to those of skill inthe art and is described in, e.g., U.S. Pat. No. 3,817,837; U.S. Pat.No. 3,850,752; U.S. Pat. No. 3,996,345 and U.S. Pat. No. 4,277,437.Other labels that will be useful include but are not limited toradioactive labels, fluorescent labels and chemiluminescent labels. U.S.Patents concerning use of such labels include for example U.S. Pat. No.3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350 and U.S.Pat. No. 3,996,345. Any of the peptides of the present invention maycomprise one, two, or more of any of these labels.

Methods of Making Specific Binding Agents

Specific binding agents of the present invention that are proteins canbe prepared by chemical synthesis in solution or on a solid support inaccordance with conventional techniques. The current limit for solidphase synthesis is about 85-100 amino acids in length. However, chemicalsynthesis techniques can often be used to chemically ligate a series ofsmaller peptides to generate full length polypeptides. Various automaticsynthesizers are commercially available and can be used in accordancewith known protocols. See, for example, Stewart and Young, Solid PhasePeptide Synthesis, 2d. ed., Pierce Chemical Co., (1984); Tam et al., JAm Chem Soc, 105:6442, (1983); Merrifield, Science, 232:341-347, (1986);and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds,Academic Press, New York, 1-284; Barany et al., Int. J. Peptide ProteinRes., 30, 705-739 (1987); and U.S. Pat. No. 5,424,398), eachincorporated herein by reference.

Solid phase peptide synthesis methods use acopoly(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g polymer.These methods for peptide synthesis use butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxy-carbonyl(FMOC) protection of alpha-amino groups.Both methods involve stepwise syntheses whereby a single amino acid isadded at each step starting from the C-terminus of the peptide (See,Coligan et al., Current Protocols in Immunology, Wiley Interscience,1991, Unit 9). On completion of chemical synthesis, the syntheticpeptide can be deprotected to remove the t-BOC or FMOC amino acidblocking groups and cleaved from the polymer by treatment with acid atreduced temperature (e.g., liquid HF-10% anisole for about 0.25 to about1 hour at 0° C.). After evaporation of the reagents, the specificbinding agent peptides are extracted from the polymer with 1% aceticacid solution that is then lyophilized to yield the crude material. Thiscan normally be purified by such techniques as gel filtration onSephadex G-15 using 5% acetic acid as a solvent. Lyophilization ofappropriate fractions of the column will yield the homogeneous specificbinding agent peptide or peptide derivatives, which can then becharacterized by such standard techniques as amino acid analysis, thinlayer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopy, molar rotation, solubility, andquantitated by the solid phase Edman degradation.

Chemical synthesis of anti-Ang-1 and/or anti-Ang-2 antibodies,derivatives, variants, and fragments thereof, as well as otherprotein-based Ang-2 binding agents permits incorporation ofnon-naturally occurring amino acids into the agent.

Recombinant DNA techniques are a convenient method for preparing fulllength antibodies and other large proteinaceous specific binding agentsof the present invention, or fragments thereof. A cDNA molecule encodingthe antibody or fragment may be inserted into an expression vector,which can in turn be inserted into a host cell for production of theantibody or fragment. It is understood that the cDNAs encoding suchantibodies may be modified to vary from the “original” cDNA (translatedfrom the mRNA) to provide for codon degeneracy or to permit codonpreference usage in various host cells.

Generally, a DNA molecule encoding an antibody can be obtained usingprocedures described herein in the Examples. Where it is desirable toobtain Fab molecules or CDRs that are related to the original antibodymolecule, one can screen a suitable library (phage display library;lymphocyte library, etc.) using standard techniques to identify andclone related Fabs/CDRs. Probes used for such screening may be fulllength or truncated Fab probes encoding the Fab portion of the originalantibody, probes against one or more CDRs from the Fab portion of theoriginal antibody, or other suitable probes. Where DNA fragments areused as probes, typical hybridization conditions are those such as setforth in Ausubel et. al. (Current Protocols in Molecular Biology,Current Protocols Press [1994]). After hybridization, the probed blotcan be washed at a suitable stringency, depending on such factors asprobe size, expected homology of probe to clone, the type of librarybeing screened, and the number of clones being screened. Examples ofhigh stringency screening are 0.1×SSC, and 0.1 percent SDS at atemperature between 50-65° C.

A variety of expression vector/host systems may be utilized to containand express the polynucleotide molecules encoding the specific bindingagent polypeptides of the invention. These systems include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransfected with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.

Mammalian cells that are useful in recombinant specific binding agentprotein productions include but are not limited to VERO cells, HeLacells, Chinese hamster ovary (CHO) cell lines, COS cells (such asCOS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293cells, as well as hybridoma cell lines as described herein. Mammaliancells are preferred for preparation of those specific binding agentssuch as antibodies and antibody fragments that are typicallyglycosylated and require proper refolding for activity. Preferredmammalian cells include CHO cells, hybridoma cells, and myeloid cells.

Some exemplary protocols for the recombinant expression of the specificbinding agent proteins are described herein below.

The term “expression vector” refers to a plasmid, phage, virus orvector, for expressing a polypeptide from a DNA (RNA) sequence. Anexpression vector can comprise a transcriptional unit comprising anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, promoters or enhancers, (2) astructural or sequence that encodes the binding agent which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription initiation and termination sequences. Structural unitsintended for use in yeast or eukaryotic expression systems preferablyinclude a leader sequence enabling extracellular secretion of translatedprotein by a host cell. Alternatively, where recombinant specificbinding agent protein is expressed without a leader or transportsequence, it may include an amino terminal methionine residue. Thisresidue may or may not be subsequently cleaved from the expressedrecombinant protein to provide a final specific binding agent product.

For example, the specific binding agents may be recombinantly expressedin yeast using a commercially available expression system, e.g., thePichia Expression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol.

The secreted specific binding agent peptide is purified from the yeastgrowth medium by, e.g., the methods used to purify the peptide frombacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding the specific binding agent peptide maybe cloned into the baculovirus expression vector pVL1393 (PharMingen,San Diego, Calif.). This vector can be used according to themanufacturer's directions (PharMingen) to infect Spodoptera frugiperdacells in sF9 protein-free media and to produce recombinant protein. Thespecific binding agent protein can be purified and concentrated from themedia using a heparin-Sepharose column (Pharmacia).

Alternatively, the peptide may be expressed in an insect system. Insectsystems for protein expression are well known to those of skill in theart. In one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) can be used as a vector to express foreign genes inSpodoptera frugiperda cells or in Trichoplusia larvae. The specificbinding agent peptide coding sequence can be cloned into a nonessentialregion of the virus, such as the polyhedrin gene, and placed undercontrol of the polyhedrin promoter. Successful insertion of the specificbinding agent peptide will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein coat. The recombinantviruses can be used to infect S. frugiperda cells or Trichoplusia larvaein which peptide is expressed [Smith et al., J Virol 46: 584 (1983);Engelhard et al., Proc Nat Acad Sci (USA) 91: 3224-7 (1994)].

In another example, the DNA sequence encoding the specific binding agentpeptide can be amplified by PCR and cloned into an appropriate vectorfor example, pGEX-3X (Pharmacia). The pGEX vector is designed to producea fusion protein comprising glutathione-S-transferase (GST), encoded bythe vector, and a specific binding agent protein encoded by a DNAfragment inserted into the vector's cloning site. The primers for thePCR can be generated to include for example, an appropriate cleavagesite. Where the specific binding agent fusion moiety is used solely tofacilitate expression or is otherwise not desirable as an attachment tothe peptide of interest, the recombinant specific binding agent fusionprotein may then be cleaved from the GST portion of the fusion protein.The pGEX-3X/specific binding agent peptide construct is transformed intoE. coli XL-1 Blue cells (Stratagene, La Jolla Calif.), and individualtransformants isolated and grown. Plasmid DNA from individualtransformants can be purified and partially sequenced using an automatedsequencer to confirm the presence of the desired specific binding agentencoding nucleic acid insert in the proper orientation.

Expression of polynucleotides encoding anti-Ang-1 and/or anti-Ang-2antibodies and fragments thereof using the recombinant systems describedabove may result in production of antibodies or fragments thereof thatmust be “re-folded” (to properly create various disulphide bridges) inorder to be biologically active. Typical refolding procedures for suchantibodies are set forth in the Examples herein and in the followingsection.

Specific binding agents made in bacterial cells may be produced as aninsoluble inclusion body in the bacteria, can be purified as follows.Host cells can be sacrificed by centrifugation; washed in 0.15 M NaCl,10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma,St. Louis, Mo.) for 15 minutes at room temperature. The lysate can becleared by sonication, and cell debris can be pelleted by centrifugationfor 10 minutes at 12,000×g. The specific binding agent-containing pelletcan be resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50%glycerol, and centrifuged for 30 min. at 6000×g. The pellet can beresuspended in standard phosphate buffered saline solution (PBS) free ofMg⁺⁺ and Ca⁺⁺. The specific binding agent can be further purified byfractionating the resuspended pellet in a denaturing SDS polyacrylamidegel (Sambrook et al., supra). The gel can be soaked in 0.4 M KCl tovisualize the protein, which can be excised and electroeluted ingel-running buffer lacking SDS. If the GST fusion protein is produced inbacteria, as a soluble protein, it can be purified using the GSTPurification Module (Pharmacia).

Mammalian host systems for the expression of the recombinant protein arewell known to those of skill in the art. Host cell strains can be chosenfor a particular ability to process the expressed protein or producecertain post-translation modifications that will be useful in providingprotein activity. Such modifications of the polypeptide include, but arenot limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Different host cells such asCHO, HeLa, MDCK, 293, WI38, as well as hybridoma cell lines, and thelike have specific cellular machinery and characteristic mechanisms forsuch post-translational activities and can be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

A number of selection systems can be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for DHFR which confers resistance to methotrexate; gptwhich confers resistance to mycophenolic acid; neo which confersresistance to the aminoglycoside G418 and confers resistance tochlorsulfuron; and hygro which that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Purification and Refolding of Specific Binding Agents

In some cases, the specific binding agents produced using proceduresdescribed above may need to be “refolded” and oxidized into a propertertiary structure and generating di-sulfide linkages in order to bebiologically active. Refolding can be accomplished using a number ofprocedures well known in the art. Such methods include, for example,exposing the solubilized polypeptide agent to a pH usually above 7 inthe presence of a chaotropic agent. The selection of chaotrope issimilar to the choices used for inclusion body solubilization, however achaotrope is typically used at a lower concentration. An exemplarychaotropic agent is guanidine. In most cases, the refolding/oxidationsolution will also contain a reducing agent plus its oxidized form in aspecific ratio to generate a particular redox potential which allows fordusykfide shuffling to occur for the formation of cysteine bridges. Somecommonly used redox couples include cysteine/cystamine,glutathione/dithiobisGSH, cupric chloride, dithiothreitol DTT/dithianeDTT, and 2-mercaptoethanol (bME)/dithio-bME. In many instances, aco-solvent may be used to increase the efficiency of the refolding.Commonly used cosolvents include glycerol, polyethylene glycol ofvarious molecular weights, and arginine.

It will be desirable to purify specific binding agent proteins orvariants thereof of the present invention. Protein purificationtechniques are well known to those of skill in the art. These techniquesinvolve, at one level, the crude fractionation of the polypeptide andnon-polypeptide fractions. Having separated the specific binding agentpolypeptide from other proteins, the polypeptide of interest can befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure specific binding agent peptide are ion-exchangechromatography, exclusion chromatography; polyacrylamide gelelectrophoresis; isoelectric focusing. A particularly efficient methodof purifying peptides is fast protein liquid chromatography or evenHPLC.

Certain aspects of the present invention concerns the purification, andin particular embodiments, the substantial purification, of an encodedspecific binding agent protein or peptide. The term “purified specificbinding agent protein or peptide” as used herein, is intended to referto a composition, isolatable from other components, wherein the specificbinding agent protein or peptide is purified to any degree relative toits naturally-obtainable state. A purified specific binding agentprotein or peptide therefore also refers to a specific binding agentprotein or peptide, free from the environment in which it may naturallyoccur.

Generally, “purified” will refer to a specific binding agent compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a specific binding agent composition inwhich the specific binding agent protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the proteins inthe composition.

Various methods for quantifying the degree of purification of thespecific binding agent will be known to those of skill in the art inlight of the present disclosure. These include, for example, determiningthe specific binding activity of an active fraction, or assessing theamount of specific binding agent polypeptides within a fraction bySDS/PAGE analysis. A preferred method for assessing the purity of aspecific binding agent fraction is to calculate the binding activity ofthe fraction, to compare it to the binding activity of the initialextract, and to thus calculate the degree of purification, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of binding activity will, of course, be dependentupon the particular assay technique chosen to follow the purificationand whether or not the expressed specific binding agent protein orpeptide exhibits a detectable binding activity.

Various techniques suitable for use in specific binding agent proteinpurification will be well known to those of skill in the art. Theseinclude, for example, precipitation with ammonium sulphate, PEG,antibodies (immunoprecipitation) and the like or by heat denaturation,followed by centrifugation; chromatography steps such as affinitychromatography (e.g., Protein-A-Sepharose), ion exchange, gelfiltration, reverse phase, hydroxylapatite and affinity chromatography;isoelectric focusing; gel electrophoresis; and combinations of such andother techniques. As is generally known in the art, it is believed thatthe order of conducting the various purification steps may be changed,or that certain steps may be omitted, and still result in a suitablemethod for the preparation of a substantially purified specific bindingagent.

There is no general requirement that the specific binding agent alwaysbe provided in its most purified state. Indeed, it is contemplated thatless substantially specific binding agent products will have utility incertain embodiments. Partial purification may be accomplished by usingfewer purification steps in combination, or by utilizing different formsof the same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low-pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of specific binding agent protein product,or in maintaining binding activity of an expressed specific bindingagent protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE [Capaldi et al.,Biochem Biophys/Res Comm, 76: 425 (1977)]. It will therefore beappreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified specificbinding agent expression products may vary.

Binding Assays

Immunological binding assays typically utilize a capture agent to bindspecifically to and often immobilize the analyte target antigen. Thecapture agent is a moiety that specifically binds to the analyte. In oneembodiment of the present invention, the capture agent is an antibody orfragment thereof that specifically binds Ang-2 and/or Ang-1. Theseimmunological binding assays are well known in the art [see, Asai, ed.,Methods in Cell Biology, Vol. 37, Antibodies in Cell Biology, AcademicPress, Inc., New York (1993)].

Immunological binding assays frequently utilize a labeling agent thatwill signal the existence of the bound complex formed by the captureagent and antigen. The labeling agent can be one of the moleculescomprising the bound complex; i.e. it can be labeled specific bindingagent or a labeled anti-specific binding agent antibody. Alternatively,the labeling agent can be a third molecule, commonly another antibody,which binds to the bound complex. The labeling agent can be, forexample, an anti-specific binding agent antibody bearing a label. Thesecond antibody, specific for the bound complex, may lack a label, butcan be bound by a fourth molecule specific to the species of antibodieswhich the second antibody is a member of. For example, the secondantibody can be modified with a detectable moiety, such as biotin, whichcan then be bound by a fourth molecule, such as enzyme-labeledstreptavidin. Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the labeling agent. These binding proteins are normalconstituents of the cell walls of streptococcal bacteria and exhibit astrong non-immunogenic reactivity with immunoglobulin constant regionsfrom a variety of species [see, generally Akerstrom, J Immunol,135:2589-2542 (1985); and Chaubert, Mod Pathol, 10:585-591 (1997)].

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures.

A. Non-Competitive Binding Assays:

Immunological binding assays can be of the non-competitive type. Theseassays have an amount of captured analyte that is directly measured. Forexample, in one preferred “sandwich” assay, the capture agent (antibody)can be bound directly to a solid substrate where it is immobilized.These immobilized antibodies then capture (bind to) antigen present inthe test sample. The protein thus immobilized is then bound to alabeling agent, such as a second antibody having a label. In anotherpreferred “sandwich” assay, the second antibody lacks a label, but canbe bound by a labeled antibody specific for antibodies of the speciesfrom which the second antibody is derived. The second antibody also canbe modified with a detectable moiety, such as biotin, to which a thirdlabeled molecule can specifically bind, such as streptavidin. [See,Harlow and Lane, Antibodies, A Laboratory Manual, Ch 14, Cold SpringHarbor Laboratory, NY (1988), incorporated herein by reference].

B. Competitive Binding Assays:

Immunological binding assays can be of the competitive type. The amountof analyte present in the sample is measure indirectly by measuring theamount of an added analyte displaced, or competed away, from a captureagent by the analyte present in the sample. In one preferred competitivebinding assay, a known amount of analyte, usually labeled, is added tothe sample and the sample is then contacted with an antibody (thecapture agent). The amount of labeled analyze bound to the antibody isinversely proportional to the concentration of analyte present in thesample. (See, Harlow and Lane, Antibodies, A Laboratory Manual, Ch 14,pp. 579-583, supra).

In another preferred competitive binding assay, the antibody isimmobilized on a solid substrate. The amount of protein bound to theantibody may be determined either by measuring the amount of proteinpresent in a protein/antibody complex, or alternatively by measuring theamount of remaining uncomplexed protein. The amount of protein may bedetected by providing a labeled protein. See, Harlow and Lane,Antibodies, A Laboratory Manual, Ch 14, supra).

Yet another preferred competitive binding assay, hapten inhibition isutilized. Here, a known analyte is immobilized on a solid substrate. Aknown amount of antibody is added to the sample, and the sample iscontacted with the immobilized analyte. The amount of antibody bound tothe immobilized analyte is inversely proportional to the amount ofanalyte present in the sample. The amount of immobilized antibody may bedetected by detecting either the immobilized fraction of antibody or thefraction that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

C. Utilization of Competitive Binding Assays:

The competitive binding assays can be used for cross-reactivitydeterminations to permit a skilled artisan to determine if a protein orenzyme complex which is recognized by a specific binding agent of theinvention is the desired protein and not a cross-reacting molecule or todetermine whether the antibody is specific for the antigen and does notbind unrelated antigens. In assays of this type, antigen can beimmobilized to a solid support and an unknown protein mixture is addedto the assay, which will compete with the binding of the specificbinding agents to the immobilized protein. The competing molecule alsobinds one or more antigens unrelated to the antigen. The ability of theproteins to compete with the binding of the specific binding agentsantibodies to the immobilized antigen is compared to the binding by thesame protein that was immobilized to the solid support to determine thecross-reactivity of the protein mix.

D. Other Binding Assays:

The present invention also provides Western blot methods to detect orquantify the presence of Ang-1 and/or Ang-2 in a sample. The techniquegenerally comprises separating sample proteins by gel electrophoresis onthe basis of molecular weight and transferring the proteins to asuitable solid support, such as nitrocellulose filter, a nylon filter,or derivatized nylon filter. The sample is incubated with antibodies orfragments thereof that specifically bind Ang-1 and/or Ang-2 and theresulting complex is detected. These antibodies may be directly labeledor alternatively may be subsequently detected using labeled antibodiesthat specifically bind to the antibody.

Binding assays to detect those Ang-1 and/or Ang-2 specific bindingagents that disrupt Ang-2 binding to its receptor are set forth in theExamples herein.

Diagnostic Assays

The antibodies or antigen-binding fragments thereof of present inventionare useful for the diagnosis of conditions or diseases characterized byexpression of Ang-1 and/or Ang-2 or subunits, or in assays to monitorpatients being treated with inducers of Ang-1 and/or Ang-2, itsfragments, agonists or inhibitors of Ang-1 and/or Ang-2 activity.Diagnostic assays for Ang-1 and/or Ang-2 include methods utilizing aspecific binding agent and a label to detect Ang-1 and/or Ang-2 in humanbody fluids or extracts of cells or tissues. The specific binding agentsof the present invention can be used with or without modification. In apreferred diagnostic assay, the specific binding agents will be labeledby attaching, e.g., a label or a reporter molecule. A wide variety oflabels and reporter molecules are known, some of which have been alreadydescribed herein. In particular, the present invention is useful fordiagnosis of human disease. A variety of protocols for measuring Ang-1and/or Ang-2 proteins using either polyclonal or monoclonal antibodiesspecific for the respective protein are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA) and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on Ang-1 and/or Ang-2 is preferred, but acompetitive binding assay can be employed. These assays are described,for example, in Maddox et al., J Exp Med, 158:1211 [1983].

In order to provide a basis for diagnosis, normal or standard values forhuman Ang-1 and/or Ang-2 expression are usually established. Thisdetermination can be accomplished by combining body fluids or cellextracts from normal subjects, preferably human, with a specific bindingagent, for example, an antibody, to Ang-1 and/or Ang-2, under conditionssuitable for complex formation that are well known in the art. Theamount of standard complex formation can be quantified by comparing thebinding of the specific binding agents to known quantities of Ang-1and/or Ang-2 protein, with both control and disease samples. Then,standard values obtained from normal samples can be compared with valuesobtained from samples from subjects potentially affected by disease.Deviation between standard and subject values suggests a role for Ang-1and/or Ang-2 in the disease state.

For diagnostic applications, in certain embodiments, specific bindingagents typically will be labeled with a detectable moiety. Thedetectable moiety can be any one that is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the detectablemoiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase, or horseradish peroxidase [Bayer et al.,Meth Enz, 184: 138-163, (1990)].

Diseases

The present invention provides a specific binding agent that binds toAng-1 and/or Ang-2 that is useful for the treatment of human diseasesand pathological conditions. Agents that modulate Ang-1 and/or Ang-2binding activity, or other cellular activity, may be used in combinationwith other therapeutic agents to enhance their therapeutic effects ordecrease potential side effects.

In one aspect, the present invention provides reagents and methodsuseful for treating diseases and conditions characterized by undesirableor aberrant levels of Ang-1 and/or Ang-2 activity in a cell. Thesediseases include cancers, and other hyperproliferative conditions, suchas hyperplasia, psoriasis, contact dermatitis, immunological disorders,and infertility.

The present invention also provides methods of treating cancer in ananimal, including humans, comprising administering to the animal aneffective amount of a specific binding agent that inhibits or decreasesAng-1 and/or Ang-2 activity. The invention is further directed tomethods of inhibiting cancer cell growth, including processes ofcellular proliferation, invasiveness, and metastasis in biologicalsystems. Methods include use of a compound of the invention as aninhibitor of cancer cell growth. Preferably, the methods are employed toinhibit or reduce cancer cell growth, invasiveness, metastasis, or tumorincidence in living animals, such as mammals. Methods of the inventionare also readily adaptable for use in assay systems, e.g., assayingcancer cell growth and properties thereof, as well as identifyingcompounds that affect cancer cell growth.

The cancers treatable by methods of the present invention preferablyoccur in mammals. Mammals include, for example, humans and otherprimates, as well as pet or companion animals such as dogs and cats,laboratory animals such as rats, mice and rabbits, and farm animals suchas horses, pigs, sheep, and cattle.

Tumors or neoplasms include growths of tissue cells in which themultiplication of the cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed malignant and may lead todeath of the organism. Malignant neoplasms or cancers are distinguishedfrom benign growths in that, in addition to exhibiting aggressivecellular proliferation, they may invade surrounding tissues andmetastasize. Moreover, malignant neoplasms are characterized in thatthey show a greater loss of differentiation (greater dedifferentiation),and of their organization relative to one another and their surroundingtissues. This property is also called “anaplasia.”

Neoplasms treatable by the present invention also include solid tumors,i.e., carcinomas and sarcomas. Carcinomas include those malignantneoplasms derived from epithelial cells that infiltrate (invade) thesurrounding tissues and give rise to metastases. Adenocarcinomas arecarcinomas derived from glandular tissue, or which form recognizableglandular structures. Another broad category or cancers includessarcomas, which are tumors whose cells are embedded in a fibrillar orhomogeneous substance like embryonic connective tissue. The inventionalso enables treatment of cancers of the myeloid or lymphoid systems,including leukemias, lymphomas and other cancers that typically do notpresent as a tumor mass, but are distributed in the vascular orlymphoreticular systems.

The type of cancer or tumor cells amenable to treatment according to theinvention include, for example, ACTH-producing tumor, acute lymphocyticleukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex,bladder cancer, brain cancer, breast cancer, cervical cancer, chroniclymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer,cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer,Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neckcancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, livercancer, lung cancer (small and non-small cell), malignant peritonealeffusion, malignant pleural effusion, melanoma, mesothelioma, multiplemyeloma, neuroblastoma, glioma, non-Hodgkin's lymphoma, osteosarcoma,ovarian cancer, ovarian (germ cell) cancer, pancreatic cancer, penilecancer, prostate cancer, retinoblastoma, skin cancer, soft tissuesarcoma, squamous cell carcinomas, stomach cancer, testicular cancer,thyroid cancer, trophoblastic neoplasms, uterine cancer, vaginal cancer,cancer of the vulva, and Wilms' tumor.

The invention is particularly illustrated herein in reference totreatment of certain types of experimentally defined cancers. In theseillustrative treatments, standard state-of-the-art in vitro and in vivomodels have been used. These methods can be used to identify agents thatcan be expected to be efficacious in in vivo treatment regimens.However, it will be understood that the method of the invention is notlimited to the treatment of these tumor types, but extends to any solidtumor derived from any organ system. Cancers whose invasiveness ormetastasis is associated with Ang-2 expression or activity areespecially susceptible to being inhibited or even induced to regress bymeans of the invention.

The invention can also be practiced by including with a specific bindingagent of the invention, such as an antibody, in combination with anotheranti-cancer chemotherapeutic agent, such as any conventionalchemotherapeutic agent. The combination of a specific binding agent withsuch other agents can potentiate the chemotherapeutic protocol. Numerouschemotherapeutic protocols will present themselves in the mind of theskilled practitioner as being capable of incorporation into the methodof the invention. Any chemotherapeutic agent can be used, includingalkylating agents, antimetabolites, hormones and antagonists,radioisotopes, as well as natural products. For example, the compound ofthe invention can be administered with antibiotics such as doxorubicinand other anthracycline analogs, nitrogen mustards such ascyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin,hydroxyurea, taxol and its natural and synthetic derivatives, and thelike. As another example, in the case of mixed tumors, such asadenocarcinoma of the breast, where the tumors includegonadotropin-dependent and gonadotropin-independent cells, the compoundcan be administered in conjunction with leuprolide or goserelin(synthetic peptide analogs of LH-RH). Other antineoplastic protocolsinclude the use of a tetracycline compound with another treatmentmodality, e.g., surgery, radiation, etc., also referred to herein as“adjunct antineoplastic modalities.” Thus, the method of the inventioncan be employed with such conventional regimens with the benefit ofreducing side effects and enhancing efficacy.

The present invention thus provides compositions and methods useful forthe treatment of a wide variety of cancers, including solid tumors andleukemias. Types of cancer that may be treated include, but are notlimited to: adenocarcinoma of the breast, prostate, and colon; all formsof bronchogenic carcinoma of the lung; myeloid; melanoma; hepatoma;neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignantcarcinoid syndrome; carcinoid heart disease; carcinoma (e.g., Walker,basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2,merkel cell, mucinous, non-small cell lung, oat cell, papillary,scirrhous, bronchiolar, bronchogenic, squamous cell, and transitionalcell); histiocytic disorders; leukemia; histiocytosis malignant;Hodgkin's disease; immunoproliferative small lung cell carcinoma;non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma;chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giantcell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma;dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma;ameloblastoma; cementoma; odontoma; teratoma; thymoma; tophoblastictumor. Further, the following types of cancers may also be treated:adenoma; cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma;cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma;hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; Sertolicell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma;myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma;ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma;neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma;paraganglioma nonchromaffin; angiokeratoma; angiolymphoid hyperplasiawith eosinophilia; angioma sclerosing; angiomatosis; glomangioma;hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma;lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma;carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma;hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma;lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma;rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervicaldysplasia.

Another aspect of the present invention is using the materials andmethods of the present invention to prevent and/or treat anyhyperproliferative condition of the skin including psoriasis and contactdermatitis or other hyperproliferative diseases. It has beendemonstrated that patients with psoriasis and contact dermatitis haveelevated Ang-2 activity within these lesions [Ogoshi et al., J. Inv.Dermatol., 110:818-23 (1998)]. Preferably, specific binding agentsspecific for Ang-2 will be used in combination with other pharmaceuticalagents to treat humans that express these clinical symptoms. Thespecific binding agents can be delivered using any of the variouscarriers through routes of administration described herein and othersthat are well known to those of skill in the art.

Other aspects of the present invention include treating variousretinopathies (including diabetic retinopathy and age-related maculardegeneration) in which angiogenesis is involved, as well asdisorders/diseases of the female reproductive tract such asendometriosis, uterine fibroids, and other such conditions associatedwith dysfunctional vascular proliferation (including endometrialmicrovascular growth) during the female reproductive cycle.

Still another aspect of the present invention relates to treatingabnormal vascular growth including cerebral arteriovenous malformations(AVMs) gastrointestinal mucosal injury and repair, ulceration of thegastroduodenal mucosa in patients with a history of peptic ulcerdisease, including ischemia resulting from stroke, a wide spectrum ofpulmonary vascular disorders in liver disease and portal hypertension inpatients with nonhepatic portal hypertension.

Another aspect of present invention is the prevention of cancersutilizing the compositions and methods provided by the presentinvention. Such reagents will include specific binding agents againstAng-2.

Pharmaceutical Compositions

Pharmaceutical compositions of Ang-1 and/or Ang-2 specific bindingagents are within the scope of the present invention. Pharmaceuticalcompositions comprising antibodies are described in detail in, forexample, U.S. Pat. No. 6,171,586, to Lam et al., issued Jan. 9, 2001.Such compositions comprise a therapeutically or prophylacticallyeffective amount of a specific binding agent, such as an antibody, or afragment, variant, derivative or fusion thereof as described herein, inadmixture with a pharmaceutically acceptable agent. In a preferredembodiment, pharmaceutical compositions comprise antagonist specificbinding agents that modulate partially or completely at least onebiological activity of Ang-1 and/or Ang-2 in admixture with apharmaceutically acceptable agent. Typically, the specific bindingagents will be sufficiently purified for administration to an animal.

The pharmaceutical composition may contain formulation materials formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.Suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates, other organic acids); bulking agents(such as mannitol or glycine), chelating agents [such as ethylenediaminetetraacetic acid (EDTA)]; complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides and other carbohydrates (such as glucose, mannose, ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring; flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counter ions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides(preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company, 1990).

The optimal pharmaceutical composition will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the specific binding agent.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute therefore. In oneembodiment of the present invention, binding agent compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the binding agent product may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery.Alternatively, the compositions may be selected for inhalation or forenteral delivery such as orally, aurally, opthalmically, rectally, orvaginally. The preparation of such pharmaceutically acceptablecompositions is within the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired specific binding agent in a pharmaceutically acceptable vehicle.A particularly suitable vehicle for parenteral injection is steriledistilled water in which a binding agent is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), beads, or liposomes, that providesfor the controlled or sustained release of the product which may then bedelivered via a depot injection. Hyaluronic acid may also be used, andthis may have the effect of promoting sustained duration in thecirculation. Other suitable means for the introduction of the desiredmolecule include implantable drug delivery devices.

In another aspect, pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

In another embodiment, a pharmaceutical composition may be formulatedfor inhalation. For example, a binding agent may be formulated as a drypowder for inhalation. Polypeptide or nucleic acid molecule inhalationsolutions may also be formulated with a propellant for aerosol delivery.In yet another embodiment, solutions may be nebulized. Pulmonaryadministration is further described in PCT Application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, binding agentmolecules that are administered in this fashion can be formulated withor without those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the binding agent molecule. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed.

Pharmaceutical compositions for oral administration can also beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

Another pharmaceutical composition may involve an effective quantity ofbinding agent in a mixture with non-toxic excipients that are suitablefor the manufacture of tablets. By dissolving the tablets in sterilewater, or other appropriate vehicle, solutions can be prepared in unitdose form. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving binding agent molecules insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. Seefor example, PCT/US93/00829 that describes controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. Additional examples of sustained-release preparationsinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate[Sidman et al., Biopolymers, 22:547-556 (1983)], poly(2-hydroxyethyl-methacrylate) [Langer et al., J Biomed Mater Res,15:167-277, (1981)] and [Langer et al., Chem Tech, 12:98-105 (1982)],ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., Proc NatlAcad Sci (USA), 82:3688-3692 (1985); EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the bindingagent molecule is being used, the route of administration, and the size(body weight, body surface or organ size) and condition (the age andgeneral health) of the patient. Accordingly, the clinician may titer thedosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical dosage may range from about 0.1 mg/kg toup to about 100 mg/kg or more, depending on the factors mentioned above.In other embodiments, the dosage may range from 0.1 mg/kg up to about100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100mg/kg.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, or pigs. An animal model may also be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

The exact dosage will be determined in light of factors related to thesubject requiring treatment. Dosage and administration are adjusted toprovide sufficient levels of the active compound or to maintain thedesired effect. Factors that may be taken into account include theseverity of the disease state, the general health of the subject, theage, weight, and gender of the subject, time and frequency ofadministration, drug combination(s), reaction sensitivities, andresponse to therapy. Long-acting pharmaceutical compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

The frequency of dosing will depend upon the pharmacokinetic parametersof the binding agent molecule in the formulation used. Typically, acomposition is administered until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose, or as multiple doses (at the same or differentconcentrations/dosages) over time, or as a continuous infusion. Furtherrefinement of the appropriate dosage is routinely made. Appropriatedosages may be ascertained through use of appropriate dose-responsedata.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional routes, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, urethral, vaginal, or rectalmeans, by sustained release systems or by implantation devices. Wheredesired, the compositions may be administered by bolus injection orcontinuously by infusion, or by implantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation of a membrane, sponge, or another appropriatematerial on to which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

In some cases, it may be desirable to use pharmaceutical compositions inan ex vivo manner. In such instances, cells, tissues, or organs thathave been removed from the patient are exposed to the pharmaceuticalcompositions after which the cells, tissues and/or organs aresubsequently implanted back into the patient.

In other cases, a binding agent which is a polypeptide can be deliveredby implanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptide. Such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic. Optionally, the cells may beimmortalized. In order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow the release of the protein product(s) but prevent the destructionof the cells by the patient's immune system or by other detrimentalfactors from the surrounding tissues.

Combination Therapy

Specific binding agents of the invention can be utilized in combinationwith other therapeutic in the treatment of Ang-1 and/or Ang-2pathologies. These other therapeutics include, for example radiationtreatment, chemotherapeutic agents, as well as other growth factors orinhibitors.

Chemotherapy treatment can employ anti-neoplastic agents including, forexample, alkylating agents including: nitrogen mustards, such asmechlorethamine, cyclophosphamide, ifosfamide, melphalan andchlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU),and semustine (methyl-CCNU); ethylenimines/methylmelamine such asthriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),hexamethylmelamine (HMM, altretamine); alkyl sulfonates such asbusulfan; triazines such as dacarbazine (DTIC); antimetabolitesincluding folic acid analogs such as methotrexate and trimetrexate,pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine,gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine,2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine,6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products includingantimitotic drugs such as paclitaxel, vinca alkaloids includingvinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine,and estramustine phosphate; ppipodophylotoxins such as etoposide andteniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin),doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin(mithramycin), mitomycinC, and actinomycin; enzymes such asL-asparaginase; biological response modifiers such as interferon-alpha,IL-2, G-CSF and GM-CSF; miscellaneous agents including platiniumcoordination complexes such as cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.

Combination therapy can done in conjunction with the growth factorslisted below or with agents that are designed to inhibit the growthfactors listed below. The growth factors include cytokines, lymphokines,growth factors, or other hematopoietic factors such as M-CSF, GM-CSF,TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF0, TNF1, TNF2,G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, anderythropoietin. Other are compositions can include known angiopoietins,for example Ang-1, -2, -4, -Y, and/or the human Ang-like polypeptide,and/or vascular endothelial growth factor (VEGF). Growth factors includeangiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bonemorphogenic protein-3, bone morphogenic protein-4, bone morphogenicprotein-5, bone morphogenic protein-6, bone morphogenic protein-7, bonemorphogenic protein-8, bone morphogenic protein-9, bone morphogenicprotein-10, bone morphogenic protein-11, bone morphogenic protein-12,bone morphogenic protein-13, bone morphogenic protein-14, bonemorphogenic protein-15, bone morphogenic protein receptor-IA, bonemorphogenic protein receptor IB, brain derived neurotrophic factor,ciliary neutrophic factor, ciliary neutrophic factor receptor,cytokine-induced neutrophil chemotactic factor-1, cytokine-inducedneutrophil, chemotactic factor-2, cytokine-induced neutrophilchemotactic factor-2, endothelial cell growth factor, endothelin-1,epidermal growth factor, epithelial-derived neutrophil attractant,fibroblast growth factor-4, fibroblast growth factor-5, fibroblastgrowth factor-6, fibroblast growth factor-7, fibroblast growth factor-8,fibroblast growth factor-8b, fibroblast growth factor-8c, fibroblastgrowth factor-9, fibroblast growth factor-10, fibroblast growth factoracidic, fibroblast growth factor basic, glial cell line-derivedneutrophic factor receptor-1, glial cell line-derived neutrophic factorreceptor-2, growth related protein, growth related protein-2, growthrelated protein-2, growth related protein-3, heparin binding epidermalgrowth factor, hepatocyte growth factor, hepatocyte growth factorreceptor, insulin-like growth factor I, insulin-like growth factorreceptor, insulin-like growth factor II, insulin-like growth factorbinding protein, keratinocyte growth factor, leukemia inhibitory factor,leukemia inhibitory factor receptor-1, nerve growth factor nerve growthfactor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor,placenta growth factor-2, platelet-derived endothelial cell growthfactor, platelet derived growth factor, platelet derived growth factor Achain, platelet derived growth factor AA, platelet derived growth factorAB, platelet derived growth factor B chain, platelet derived growthfactor BB, platelet derived growth factor receptor-1, platelet derivedgrowth factor receptor-2, pre-B cell growth stimulating factor, stemcell factor, stem cell factor receptor, transforming growth factor-1,transforming growth factor-2, transforming growth factor-3, transforminggrowth factor-1.2, transforming growth factor-4, transforming growthfactor-5, latent transforming growth factor-1, transforming growthfactor binding protein I, transforming growth factor binding protein II,transforming growth factor binding protein III, tumor necrosis factorreceptor type I, tumor necrosis factor receptor type II, urokinase-typeplasminogen activator receptor, vascular endothelial growth factor, andchimeric proteins and biologically or immunologically active fragmentsthereof.

Combination therapy can also be achieved with a specific binding agentof the present invention, such as an antibody, in combination with anapoptotic inducer such as a specific binding agent (e.g., an agonisticantibody or TRAIL ligand) that induces apoptosis via the DR4 (TRAIL R-1)and/or the DR5 (TRAIL R-2) receptor. Examples of such specific bindingagents are provided in WO 2007/027713, incorporated herein by reference,which discloses agonistic antibodies that induce apoptosis via the DR5receptor.

Immunotherapeutics

Immunotherapeutics generally rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectorsmay be, for example an antibody of the present invention that recognizessome marker on the surface of a target cell. The antibody alone mayserve as an effector of therapy or it may recruit other cells toactually effect cell killing. The antibody may also be conjugated to adrug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and thus may merely serve as a targetingagent.

According to the present invention, mutant forms of Ang-1 and/or Ang-2may be targeted by immunotherapy either antibodies or antibodyconjugates of the invention. It is particularly contemplated that theantibody compositions of the invention may be used in a combined therapyapproach in conjunction with Ang-2 targeted therapy.

Passive immunotherapy has proved to be particularly effective against anumber of cancers. See, for example, WO 98/39027.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

Example 1 Generation of Affinity Matured Antibodies Against Ang2 byPhage Display Overall Strategy

CDR randomization was employed to enhance the activity of Ang2 antibody,similar to previous approaches (Chen Y et al., 1999 J Mol Biol(293)865-881; Yelton D E et al., 1995 J Immunol (155) 1994-2004; YangW-P et al., 1995 J Mol Biol (254) 392-403). Briefly, the variableregions of Ang2 antibody 536 were cloned into the TargetQuest modifiedpCES-1 vector (Dyax Corp, de Haard H J et al 1999 J Biol Chem (274)18218-30). All CDR regions were targeted for randomization of each CDRresidue by mutagenesis using NNK containing oligonucleotides. After themutagenesis reaction, phage clones were interrogated for each positionusing phage ELISA to identify beneficial mutations (for methods see WO2004/046306,WO 2003/03057134, and US 2003/0099647 A1, for general phageantibody refs., Marks J D et al., 1991 J Mol Biol (222) 581-597;Hoogenboom H R et al 1992 J Mol Biol (227) 381-388; Griffiths A D etal., 1993 EMBO J. (12) 725-734; Vaughan T P et al., 1996 Nat Biotechnol(14) 309-314). Clones with beneficial mutations were converted to fullantibodies. Heavy chain clones were paired with light chain clones andresulting IgG was tested for neutralization activity. Top 22 clones werecharacterized further.

A. Ab536 Fab Template Construction

The variable regions of 536 antibody were cloned into pCES-1 vectorusing standard molecular biology techniques (Molecular Cloning: ALaboratory Manual, 3^(rd) Edition Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 2001). The heavy chain variable and fulllength light chain fragments were generated by PCR using the followingoligonucleotides:

Heavy chain reverse: CCGCTGTGCCCCCAGAGGTGC Heavy chain forward:ttttttccatggccgaggtccagctggtgcagtc Light chain reverse:TTTTTTGGCGCGCCTTATTAACACTCTCCCCTGTTGAAGCT Light chain forward:ttttttgtgcacttgacattgtgatgactcagtct

The variable region of heavy chain was inserted between the restrictionsites, NcoI and BstEII. The full length light chain was inserted betweenrestriction sites, ApaLI and AscI. The resulting construct was used as atemplate for CDR randomization.

B. CDR Mutations

Antibody 536 as a Fab in vector pCES-1 was affinity matured by astep-wise site-directed mutagenesis using oligonucleotides bearing NNK(N=ATCG; K=GT) codons for each of the CDR positions. The QuikChangeSite-Directed Mutagenesis Kit (Stratagene #200518-5) was used followingthe manufacturer recommended protocol. To identify phages with enhancedbinding to Ang2, phage ELISA performed with biotinylated human Ang2protein coated at 2 ug/ml in PBS onto the 96 well Maxisorp plates(NUNC). Briefly, after blocking with 2% milk in PBS, overnight phageculture that were grown with helper phage was incubated and bound phageswere detected with anti-M13 antibodies conjugated with HRP (Pharmacia).Luminescence signal was compared relative to parental 536 Fab, andclones with superior signal were selected for further analysis.

C. IgG Conversion of Phage Fab

After phage ELISA and sequence analysis, 95 clones each from light chainand heavy chain mutagenesis with enhanced binding against Ang2 wereselected and converted into IgG. Briefly, the variable regions of eachclone were PCR amplified using a pair of primers. Primer sequences forLC were CTG CTG CTG TGG CTG AGA GGT GCG CGC TGT GAT ATT GTG ATG ACT CAGTCT CCA CTC TCC and AAA AAA CGT ACG TTT GAT CTC CAG CTT GGT CC. Primersfor HC were TTTTTTTTGCGCGCTGTGAGGTCCAGCTGGTGCAGTC and AAAAAAGGCACTAGAGACGGTGACCAGGGTTCC. After digesting with BssHII and BsiWI for LC, andBssHII and BsmBI for HC, the variable regions were inserted into pcDNA3vectors containing VK1 leader sequence and constant sequence of humanKappa and human IgG1 using standard molecular biology techniques. Eachligation mixture was transformed into two 96 well plates of XL10 goldcompetent cells (Stratagene), and the transformation mixture were grownovernight for plasmid prep the next day. Resulting DNA were paired withrelative parental 536 LC or HC DNA, and transiently transfected into293T cells in OPTI-MEM using Fugene6. After 7 days, the media fromtransfected cells were collected, and IgG concentration was quantifiedby Lance assay using anti-human IgG antibody (Fc specific) labeled witheuropium and anti-human IgG antibody labeled with APC (Perkin Elmer).

D. Selection of IgG Clones

Conditioned media that contain IgG were tested in HTRF neutralizingassay for its inhibitory effect of Tie-2 interaction with either Ang1 orAng2. From initial screening, 15LC clones and 11HC clones that showedimproved activity were picked. The DNA of selected clones were preparedand confirmed by sequencing. Then the combination of each LC and HCmutants, along with 536 parental clone, were transfected into two 96plates seeded with 293T cells. Conditioned media were collected, andanalyzed for the IgG concentration and inhibitory effect in Tie2neutralizing assay. From this combination, 22 clones that contain singlemutation in each LC and HC were selected for further analysis.

E. Expression and Purification of Human Affinity Matured Ang2 Antibodiesin CHO Cells

CS-9 cells used for transfection of the anti-Ang2 IgG expressionplasmid(s) are a serum-free suspension CHO cell line. They were derivedby gradually adapting DXB-11 CHO cells to grow in serum-free medium asdescribed in Rasmussen et al, 1998 (Rasmussen, B., Davis, R., Thomas,J., Reddy, P. 1998. Isolation, characterization and recombinant proteinexpression in Veggie-CHO: A serum-free CHO host cell line.Cytotechnology. 28: 31-42). DXB-11 cells are a DHFR-deficient mutantderivative from CHO-K1 cells. (Chasin and Urlaub, 1983; Urlaub andChasin. 1980. Isolation of Chinese hamster cell mutants deficient indihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77,4216-4220.; Chasin L. A., Graf, L., Ellis, N., Lanzberg, M., Urlaub, G.1982. Gene amplification in dihydro folate reductase deficient mutants.Schimke, R. T. (Ed.) Gene amplification; Cold Spring Harbor, N.Y. ColdSpring Harbor Laboratory: Cold Spring Harbor, N.Y., p161-166). CHO-K1 isan epithelioid cell line originally isolated from the Chinese hamsterovary (Kao and Puck. Genetics of somatic mammalian cells. VII. Inductionand isolation of nutritional mutants in Chinese hamster cells. Proc.Nat. Acad. Sci. 60: 1275-1281, 1968).

To derive the CS-9 host cell line, DXB-11 cells were grown in media withgradual reduction in serum over 100 passages to obtainserum-free-adapted cells referred to as SF-CHO (Rasmussen et al, 1998).The SF-CHO cells were subsequently sub-cloned by limiting dilutioncloning and individual clones were evaluated. The CS-9 clone wasselected as the host cell line for expression of recombinant proteinsand banked in serum-free medium. The bank was tested for adventiousagents and sterility and found to be free of viral, mycoplamsa andmicrobial agents. The host cell line, CS-9, is a DHFR deficient CHO cellline auxotrophic for glycine, hypoxanthine and thymidine (GHT). Theplasmids pDC323 and pDC324 each encode a portion of the DHFR cDNA andthe 2 plasmids must complement each other to express a functional DHFRmolecule by association of the 2 DHFR fragments in vivo.

The following twenty-two antibodies, each consisted of two heavy chainsand 2 light (kappa or lambda) chains as designated in the followingTable 2.

TABLE 2 Antibody Heavy Antibody Light Antibody* Chain Chain H6L7 H6 HCL7 LC H5L7 H5 HC L7 LC H4L13 H4 HC L13 LC H11L7 H11 HC L7 LC H10L7 H10HC L7 LC H4L7 H4 HC L7 LC H5L6 H5 HC L6 LC H2L7 H2 HC L7 LC H5L8 H5 HCL8 LC H6L8 H6 HC L8 LC H3L7 H3 HC L7 LC H5L4 H5 HC L4 LC H4L12 H4 HC L12LC H6L6 H6 HC L6 LC H4L2 H4 HC L2 LC H4L6 H4 HC L6 LC H4L4 H4 HC L4 LCH5L11 H5 HC L11 LC H5L1 H5 HC L1 LC H4L11 H4 HC L11 LC H5L12 H5 HC L12LC H5L9 H5 HC L9 LC *Tested for binding to hAng-2, mAng-2, and hAng-1 asdescribed herein.Tables 3 and 4 set forth the sequences and SEQ ID NOs. of the heavy andlight (kappa and lambda) chains of the 22 anti-Ang-1 and/or anti-Ang-2antibodies converted from phage to full length IgG1 antibodies. Thecomplementarity-determining regions (CDRs) of the monoclonal antibodieswere predicted using the VBASE database which uses the techniquedescribed by Kabat et al in: Sequences of Proteins of ImmunologicalInterest (NIH Publication No. 91-3242; U.S. Dept. Health and HumanServices, 5^(th) ed.). Fab regions were aligned to sequences in thedatabase with the closest germline sequence and then visually comparedwith such sequences. The CDRs for each variable region (heavy or lightchain), both residue and sequences are set forth in Table 5.

TABLE 3 Heavy Chain Variable Regions Antibody HC Sequence 536 HCEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (Ref)GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS H2EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 1)GLEWVSYISSSGSTIEYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS H3EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 2)GLEWVSYISSSGSTIQYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS H4EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 3)GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDLLTGYGYWGQGTLVTVSS H6EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 4)GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDIYTGYGYWGQGTLVTVSS H10EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO 5)GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGLWGQGTLVTVSS H11EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 6)GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGMWGQGTLVTVSS H5PEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 7)GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDIWTGYGYWGQGTLVTVSS

TABLE 4 Light Chain Variable Regions Antibody LC Sequence 536 kappaDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (Ref)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L1DIVMTQSPLSLPVTPGEPASISCRSIQSLLQSNGYNYLDWYLQKP (SEQ ID NO. 8)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L2DIVMTQSPLSLPVTPGEPASISCRSSQSLLLSNGYNYLDWYLQKP (SEQ ID NO. 9)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L4DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSHGYNYLDWYLQKP (SEQ ID NO. 10)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L6DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSVGYNYLDWYLQKP (SEQ ID NO. 11)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L7DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKP (SEQ ID NO. 12)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L8DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNMLDWYLQKP (SEQ ID NO. 13)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L9DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 14)GQSPQLLIYAGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L11DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 15)GQSPQLLIYLGSDRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPPTFGQGTKLEIK L12DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 16)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQATHWPPTFGQGTKLEIK L13DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 17)GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQVTHWPPTFGQGTKLEIK

TABLE 5Complementarity-Determining Regions (CDRs) of Heavy Chains (HC) andLight Chains (LC) of Ang-1 and/or Ang-2 Antibodies; Residues andSequence CDR 1 CDR 2 CDR 3 Antibody Residues Sequence Residues SequenceResidues Sequence Ab 536 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDILTGYGY HC Ab 536 24-39 RSSQSLLHSNGYNYLD 55-61 LGSNRAS 94-102MQGTHWPPT LC H6L7 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 32) H6L7 LC 24-39RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19) (SEQ ID NO. 27)(SEQ ID NO. 33) H5L7 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L7 LC 24-39RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19) (SEQ ID NO. 27)(SEQ ID NO. 33) H4L13  31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYD

LTGYGY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L13  24-39RSSQSLLHSNGYNYLD 55-61 LGSNRAS 94-102 MQ

THWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 27) (SEQ ID NO. 36) H11L7  31-35SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDILTG

GY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 37) H11L7  24-39RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT LC (SEQ ID NO. 19) (SEQ ID NO. 27)(SEQ ID NO. 33) H10L7  31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDILTG

GY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 38) H10L7  24-39RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT LC (SEQ ID NO. 19) (SEQ ID NO. 27)(SEQ ID NO. 33) H4L7 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYD

LTGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L7 LC 24-39RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19) (SEQ ID NO. 27)(SEQ ID NO. 33) H5L6 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L6 LC 24-39RSSQSLLHS

GYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 21) (SEQ ID NO. 27)(SEQ ID NO. 33) H2L7 HC 31-35 SYGMH 50-66 YISSSGSTI

YADSVKG 99-111 DLLDYDILTGYGY (SEQ ID NO. 18) (SEQ ID NO. 28) (SEQ IDN0. 39) H2L7 LC 24-39 RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19) (SEQ ID NO. 27)(SEQ ID NO. 33) H5L8 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L8 LC 24-39RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 22) (SEQ ID NO. 27)(SEQ ID NO. 33) H6L8 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 32) H6L8 LC 24-39RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 22) (SEQ ID NO. 27)(SEQ ID NO. 33) H3L7 HC 31-35 SYGMH 50-66 YISSSGSTI

YADSVKG 99-111 DLLDYDILTGYGY (SEQ ID NO. 18) (SEQ ID NO. 29) (SEQ IDN0. 39) H3L7 LC 24-39 RSSQSLLHSNGYN

LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19) (SEQ ID NO. 27)(SEQ ID NO. 33) H5L4 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L4 LC 24-39RSSQSLLHS

GYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 23) (SEQ ID NO. 27)(SEQ ID NO. 33) H4L12  31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYD

LTGYGY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L12  24-39RSSQSLLHSNGYNYLD 55-61 LGSNRAS 94-102 MQ

THWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 27) (SEQ ID NO. 40) H6L6 HC 31-35SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 32) H6L6 LC 24-39RSSQSLLHS

GYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 21) (SEQ ID NO. 27)(SEQ ID NO. 33) H4L2 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYD

LTGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L2 LC 24-39RSSQSLL

SNGYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 24) (SEQ ID NO. 27)(SEQ ID NO. 33) H4L6 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYD

LTGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L6 LC 24-39RSSQSLLHS

GYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 21) (SEQ ID NO. 27)(SEQ ID NO. 33) H4L4 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYD

LTGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L4 LC 24-39RSSQSLLHS

GYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 23) (SEQ ID NO. 27)(SEQ ID NO. 33) H5L11  31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111DLLDYDI

TGYGY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L11  24-39RSSQSLLHSNGYNYLD 55-61 LGS

RAS 94-102 MQGTHWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 30) (SEQ ID NO. 33)H5L1 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L1 LC 24-39 RS

QSLL

SNGYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 25) (SEQ ID NO. 27)(SEQ ID NO. 33) H4L11  31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYD

LTGYGY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L11  24-39RSSQSLLHSNGYNYLD 55-61 LGS

RAS 94-102 MQGTHWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 30) (SEQ ID NO. 33)H5L12  31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI

TGYGY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L12  24-39RSSQSLLHSNGYNYLD 55-61 LGSNRAS 94-102 MQ

THWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 27) (SEQ ID NO. 40) H5L9 HC 31-35SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI

TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L9 LC 24-39RSSQSLLHSNGYNYLD 55-61

GSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 20) (SEQ ID NO. 31) (SEQ ID NO. 33)

Example 2 Molecular Assays to Evaluate Ang-2 Antibodies

Molecular assays (Affinity ELISA, Neutralization ELISA and BIAcore) weredeveloped to assess direct antibody binding to Ang-2 and related familymembers (for example, Ang-1), and the effect of antibodies on theAng-2:Tie2 interaction. These in vitro and cell-based assays aredescribed as follows.

A. Affinity ELISA

For the initial screening of candidate anti-Ang-2 antibodies, purifiedhuman Ang-2 (R and D Systems, Inc; catalog number 623-AN; Ang-2 isprovided as a mixture of 2 truncated versions) or murine Ang-2polypeptide (prepared as described above) were used. For confirmatorybinding assays, human Ang-2 was obtained from conditioned media of human293T cells transfected with full length human Ang-2 DNA and cultured inserum free DMEM containing about 50 micrograms per ml of bovine serumalbumin (BSA).

Using microtiter plates, approximately 100 microliters per well of Ang-2was added to each well and the plates were incubated about 2 hours,after which the plates were washed with phosphate buffered saline (PBS)containing about 0.1 percent Tween-20 four times. The wells were thenblocked using about 250 microliters per well of about 5 percent BSA inPBS, and the plates were incubated at room temperature for about 2hours. After incubation, excess blocking solution was discarded, andabout 100 microliters of candidate anti-Ang-2 antibody was added to eachwell in a dilution series starting at a concentration of about 40nanomolar and then serially diluting 4-fold in PBS containing about 1percent BSA. The plates were then incubated overnight at roomtemperature. After incubation, plates were washed with PBS containingabout 0.1 percent Tween-20. Washing was repeated four additional times,after which about 100 microliters per well of goat anti-humanIgG(Fc)-HRP (Pierce Chemical Co., catalog #31416) previously diluted1:5000 in PBS containing 1 percent BSA (bovine serum albumin) was added.Plates were incubated approximately 1 hour at room temperature. Plateswere then washed five times in PBS containing about 0.1 percentTween-20, after which about 100 microliters per well of TMB(3,3′,5,5′-Tetramethylbenzidine Liquid Substrate System; Sigma chemicalCompany, St. Louis, Mo., catalog number T8665) substrate was added andplates were incubated about 5-15 minutes until blue color developed.Absorbance was then read in a spectrophotomer at about 370 nm.

B. Neutralization ELISA

Microtiter plates to which human Ang-2 polypeptide was bound wereprepared as described for the Affinity ELISA. Candidate anti-Ang-2antibodies were prepared in serial dilutions as described for theAffinity ELISA above in a solution of PBS containing about 1 percent BSAand about 1 nM Tie2 (provided as a Tie2-Fc molecule where the Tie2portion contains only the soluble extracellular portion of the molecule;R and D Systems, catalog number 313-TI). After about 100 microliters ofthe antibody/Tie2 solution was added to each well, the plates wereincubated overnight at room temperature, and then washed five times inPBS containing about 0.1 percent Tween-20. After washing, about 100microliters per well of anti-Tie2 antibody (Pharmingen Inc., catalog#557039) was added to a final concentration of about 1 microgram per mland the plates were incubated about 1 hour at room temperature thenwashed five time in PBS containing about 0.1 percent Tween-20. Next,about 100 microliters per well of goat anti-mouse-IgG-HRP (PierceChemical CO., catalog #31432) was added at a dilution of 1:10,000 in PBScontaining about 1 percent BSA. Plates were incubated at roomtemperature for about 1 hour, after which they were washed five timeswith PBS containing about 0.1 percent Tween-20. About 100 microlitersper well of TMB substrate (described above) was then added and color wasallowed to develop. Absorbance was then read in a spectrophotomer at 370nm.

C. Affinity BIAcore

An affinity analysis of each candidate Ang-2 antibody was performed on aBIAcore®2000 (Biacore, Inc., Piscataway, N.J.) with PBS and 0.005percent P20 surfactant (BIAcore, Inc.) as running buffer. RecombinantProtein G (Repligen, Needham, Mass.) was immobilized to a research gradeCM5 sensor chip (Biacore, Inc.) via primary amine groups using the AmineCoupling Kit (Biacore, Inc.) according to the manufacturer's suggestedprotocol.

Binding assays were carried out by first attaching about 100 Ru of eachcandidate anti-Ang-2 antibody to the immobilized Protein G, after whichvarious concentrations (0-100 nM) of huAng-2 or mAng-2 were theninjected over the bound antibody surface at a flow rate of about 50ul/min for about 3 minutes. Antibody binding kinetics including k_(a)(association rate constant), k_(d) (dissociation rate constant) andK_(D) (dissociation equilibrium constant) were determined using the BIAevaluation 3.1 computer program (BIAcore, Inc.). Lower dissociationequilibrium constants indicated greater affinity of the antibody forAng-2.

All twenty two of the antibodies and a negative control IgG1 (referredto as RDB1) were tested using affinity and neutralization ELISA (asdescribed in Example 3 above) as well as the BIAcore neutralizationassay to determine their affinity, neutralization, and specificitycapabilities. The results are set forth below (Table 2) and werecalculated using standard procedures. Three antibodies, H6L7, H4L4 andH4L11 were evaluated for IC50 neutralizing concentrations against humanand murine Ang-1 and Ang-2., using the ELISA analysis described above.All three antibodies were shown to crossreact with mouse, rabbit, andcynomolgus monkey Ang1 and Ang2, exhibiting similar potencies acrossangiopoietin orthologs. The results are reported in the following Table3.

D. HTRF hAng-1 and hAng-2 Antibody IC50s and IC90s

Equal volume of 1.6 nM Streptavidin-Europium (SA-EU) and 8 nMBiotinylated angiopoietin 2 (in-house) or Biotinylated agiopoietin 1(R&D Cat# BAF923) were mixed and incubated at room temperature for 30minutes in the dark with rotation in a 15 ml conical tube (Fisher352096). Then 50 ul of the above SA-EU/Biotinylated Ang 2(1) mixture wasadded to each well on a Mixing Plate (Costar 3356). To the Mixing Plate,50 ul of serial diluted Ang1 and Ang2 antibody at 4× finalconcentrations were added to each well. The Mixing Plate was thenincubated at room temperature for 1 hour on a shaker in the dark. On anAssay Plate (Costar 3356), 20 ul of 10 nM huTie-2-Fc-APC (Prozyme CustomLot# DF99-048) was added to each well. Then 20 ul of the mixture fromeach well on the Mixing Plate was transferred to each well on the AssayPlate. The Assay Plate was incubated at room temperature for 2 hours inthe dark with rotation. Then the Assay Plate was read on RUBYstar platereader (BMG labtechnologies, INC). All the reagents in the assay werediluted with HTRF buffer (50 mM Tris HCl, 100 mM NaCl, 0.1% BSA and0.05% Tween 20). IC50 and IC90 were calculated with GRAFIT 5.0.

TABLE 6 Biochemical Potency of Antibodies Against hAng1 and hAng2 IC50IC90 IC50 IC90 hAng1 hAng1 IC90/ hAng2 hAng2 IC90/ Antibody

IC50

IC50 H6L7 0.06 0.49 8.0 0.06 0.19 3.3 H5L7 0.07 0.42 6.3 0.07 0.23 3.6H4L13 0.15 1.6 11 0.06 0.19 3.2 H11L7 0.15 1.2 8.1 0.06 0.20 3.2 H10L70.15 2.2 14 0.06 0.19 3.4 H4L7 0.23 2.8 12 0.06 0.22 3.5 H5L6 0.32 3.611 0.07 0.23 3.4 H2L7 0.33 3.6 11 0.06 0.20 3.3 H5L8 0.37 3.6 10 0.070.21 3.1 H6L8 0.57 7.7 13 0.05 0.19 3.6 H3L7 0.58 7.1 12 0.06 0.23 4.0H5L4 0.60 11 19 0.07 0.21 2.8 H4L12 0.63 8.7 14 0.06 0.21 3.4 H6L6 0.6610 16 0.06 0.20 3.4 H4L2 0.66 6.8 10 0.06 0.19 3.1 H4L6 0.74 15 20 0.060.20 3.2 H4L4 0.87 8.2 9.4 0.06 0.16 2.7 H5L11 0.97 18 18 0.08 0.25 3.3H5L1 1.7 24 15 0.06 0.29 4.4 AMG 386* 2.6 106 41 0.03 0.13 4.1 AMG 386*3.9 278 71 0.03 0.15 4.4 H4L11 7.3 107 15 0.05 0.17 3.4 H5L12 14 159 110.07 0.31 4.4 H5L9 18 181 10 0.19 1.57 8.5 *Peptibody.

indicates data missing or illegible when filed

Example 3 Molecular Characterization of Angiopoietin Antibodies

Four of the fully human IgG2 antibodies (Ab536, H4L4, H6L7, and H4L11)with potent hAng2 inhibitory activity and a range of hAng1 inhibitoryactivities were selected for further studies. All 4 antibodies wereshown to crossreact with mouse, rabbit, and cynomolgus monkey Ang1 andAng2, exhibiting similar potencies across angiopoietin orthologs (Tables7 and 8).

TABLE 7 Biochemical Potency of Angiopoietin Antibodies Against Ang2Orthologs Human Ang2 Cyno Ang2 Murine Ang2 Rabbit Ang2 Clone IC50 (nM)IC50 (nM) IC50 (nM) IC50 (nM) H6L7 0.22 0.19 0.13 0.15 H4L4 0.22 0.240.15 0.15 AMG 386 0.12 0.17 0.10 0.10 H4L11 0.21 0.16 0.12 0.12 536 LC10.27 0.19 0.14 0.20ELISA measuring neutralization of ligand/receptor interaction.

TABLE 8 Biochemical Potency of Angiopoietin Antibodies Against Ang1Orthologs Human Ang1 Cyno Ang1 Murine Ang1 Rabbit Ang1 Clone IC50 (nM)IC50 (nM) IC50 (nM) IC50 (nM) H6L7 0.12 0.19 0.12 0.18 H4L4 3.2 4.0 3.32.8 AMG 386 1.2 2.9 2.7 4.3 H4L11 17 14 7.7 16 536 LC1 515 531 305 502ELISA measuring neutralization of ligand/receptor interaction.

Example 4 Activity of Angiopoietin Antibodies in Colo205 TumorXenografts

Three antibodies (H6L7, H4L4 and H4L11) were evaluated in the Colo205human colorectal carcinoma xenograft model. For each study group, micewere injected subcutaneously on the right flank with 2×106 cells inMatrigel™. Ten animals with average tumor volume of 300 mm3 wererandomly assigned to each experimental group. The animals were injectedIP twice per week, beginning on day 17 post implantation, with 300 μg ofthe angiopoietin-targeted antibodies or isotype control antibody. AMG386 at the optimum biological dose (OBD) in this model of 14 μg (SC)twice weekly was included as a positive control and antibody 536LC1 wasincluded at 300 μg twice weekly. Body weight and tumor size weremeasured twice weekly.

As shown in FIG. 1, all three antibodies significantly inhibited tumorgrowth compared to treatment with an isotype control antibody(p<0.0001). Treatment with H6L7 and H4L4, resulted in significantlygreater inhibition of tumor growth compared to 536LC1. Data representsmean±SEM. At the end of the experiment, tumors were harvested, fixed inzinc-formalin and paraffin embedded. Histological sections of tumor werestained with hematoxylin. The viable tumor fraction was then estimated,using RGB thresholding and automated pixel counting, from a 1× digitalimage of the entire tumor cross-section. Viable tumor burden wascalculated as the viable fraction multiplied by the terminal tumorweight. Data represents mean±SEM (n=10). FIG. 2 demonstrates thatantibodies H6L7, H4L4 and H4L11 also significantly reduced tumor burdenrelative to control (p<0.0001), suggesting that the volume-based tumormeasurements underestimated the anti-tumor effect of the antibodies.

Histopathology was performed on tumors and normal tissues from the micein the study shown in FIGS. 1 and 2. Treatment of xenograft-bearing nudemice with the angiopoietin inhibitors (AMG 386, 536LC1, H4L4, H4L11, orH6L7) did not elicit adverse anatomic effects in non-target tissues.

Example 5 Effect of Anti-Ang-1 and/or Ang-2 Antibodies on EndothelialCell Proliferation

In a parallel experiment, animals with approximately 400 mm³ tumors weretreated with 536LC1 (AKA LC1), H4L11, H4L4, H6L7, AMG 386 or controlIgG2 for 72 hrs. Seventeen hours prior to sacrifice, animals wereimplanted with osmotic minipumps containing 3 mg/mL BrdU. Uponsacrifice, endothelial cells were isolated from the Colo205tumor-bearing mice and were analyzed by flow cytometry to assessproliferation. Dissociated cells were stained with anti-mouse CD45-FITCand CD31-PE antibodies, followed by fixation and staining withanti-BrdU-alexa647 antibodies. Data represents mean±SEM (n=5). As shownin FIG. 3, treatment with all antibodies significantly reduced thepercentage of BrdU positive cells (p<0.002 compared to IgG2 control).These data are consistent with an anti-angiogenic therapeutic mechanismwhereby the angiopoietin-targeted antibodies inhibit tumor endothelialcell proliferation in vivo.

Example 6 Dose Titration of H4L4 in Colo205 Tumor Xenografts

The antibody H4L4 was selected for more extensive analysis exploring thedose dependency of H4L4-mediated tumor growth inhibition. The animalswere injected with H4L4 IP twice-weekly beginning on day 14 at dosesranging from 3 μg to 300 μg. AMG 386 at the optimum biological dose of14 μg (SC) twice weekly was included as a positive control. As shown inFIG. 4, all doses of H4L4 significantly inhibited tumor growth andviable tumor burden (p<0.0001), with an OBD of ˜30 μg in the viabletumor burden analysis (FIG. 5).

Example 7 Effect of H4L4 on Colo205 Tumor Endothelial Cell ProliferationIn Vivo

In a parallel experiment, Colo205 tumor-bearing mice with tumors ofapproximately 450 mm³ were treated with a single dose of H4L4, AMG 386or control IgG2 for 72 hours and then analyzed as in FIG. 3. As shown inFIG. 6, treatment with H4L4 significantly inhibited endothelial cellproliferation in a dose-dependent manner, with an OBD of 30 μg.

Example 8 Pharmacokinetics of H4L4, H6L7, H4L11, and 536LC1 in Mice,Rats and Cynomologus monkey

The pharmacokinetics (PK) of H4L4, H6L7, H4L11, and 536LC1 have beencharacterized in CD-1 mice after single-dose intravenous (IV) orintraperitoneal (IP) administration. The PK of H4L4 and H6L7 was alsocharacterized in Sprague-Dawley rats and cynomolgus monkeys aftersingle-dose IV administration.

After single-dose IV or IP administration to mice, H4L4 exposureappeared to increase approximately dose-proportionally in the dose rangeof 0.1 to 10 mg/kg (Table 4). The overall mean terminal half-life(t_(1/2,z)), clearance (CL), and volume of distribution at steady-state(V_(ss)) was 207 hrs, 0.43 mL/hr/kg, and 128 mL/kg, respectively. Thebioavailability (% F) after IP administration was greater than 90% forall dose groups. In contrast, H6L7, H4L11, and 536LC1 exhibitednonlinear PK in mice with exposure increasing greater than doseproportionally from 0.1 to 10 mg/kg. The exposure of H6L7 in rats andmonkeys also increased greater than dose-proportionally after a singleIV dose of 0.1 to 10 mg/kg.

In contrast to its linear PK profile in mice, H4L4 exhibited nonlinearrat and monkey PK. The mean residence time (MRT) in rats ranged from 57to 217 hours; the CL ranged from 0.3 to 1.4 mL/hr/kg; the V_(ss) rangedfrom 57 to 68 mL/kg. In monkeys, the MRT ranged from 40 to 163 hours;the CL ranged from 0.4 to 1.9 mL/hr/kg; the V_(ss) ranged from 49 to 75mL/kg.

The PK of H4L4 was also assessed in nude mice bearing Colo205 tumorxenografts in a pharmacology study at 3, 10, 30, 100 or 300 μgdose/mouse, administered IP twice weekly for 4 weeks. Serum H4L4exposure increased approximately dose proportionally as assessed byserum trough concentrations. The PK of H4L4 in nude mice was similar tothat observed in CD-1 mice, and PK did not appear to change over time.

TABLE 9 PK Parameters of H4L4 and H6L7 in Preclinical Species Mouse RatMonkey 0.1 1 10 0.1 1 10 0.1 1 10 H4L4 Dose (mg/kg) t_(1/2,z) (hr) 196180 244 42.5 66.7 213 36.2 35.6 80.1 MRT (hr) 259 273 420 57.4 107 21740.1 52.0 163 CL (mL/hr/kg) 0.666 0.386 0.249 1.39 0.552 0.319 1.890.933 0.414 V_(ss) (mL/kg) 173 105 105 66.4 57.0 68.5 74.9 48.6 67.4 V₀(mL/kg) 71.0 46.6 46.7 43.3 34.5 36.1 48.2 40.8 44.0 H6L7 Dose (mg/kg)t_(1/2,z) (hr) 8.27 99.0 82.8 9.43 25.8 92.9 10.4 32.6 65.0 MRT (hr)11.6 54.3 263 13.2 41.2 158 15.1 51.8 158 CL (mL/hr/kg) 19.4 2.00 0.3493.31 1.02 0.331 2.81 0.824 0.358 V_(ss) (mL/kg) 224 109 91.8 43.3 41.351.9 41.3 42.2 55.2 V₀ (mL/kg) 90.9 58.4 56.6 35.4 35.3 38.1 40.0 37.743.1

Example 9 Angiopoietin-1 Neutralization Mediates Context-DependentSuppression of Angiogenesis and Tumor Growth

While Angiopoietin-2 (Ang2) is a key mediator of postnatal angiogenesis,the role of Angiopoietin-1 (Ang1) in this setting is less clear. Toinvestigate the postnatal function of Ang1, we have developed potent andselective peptibodies (peptide-Fc fusion proteins) that inhibit theinteraction between Ang1 and its receptor, Tie2. We show that selectiveAng1 antagonism has no independent effect in models ofangiogenesis-associated diseases (cancer and diabetic retinopathy),although it can induce ovarian atrophy in normal juvenile rats andinhibit ovarian follicular angiogenesis in a hormone-induced ovulationmodel. Surprisingly, the activity of Ang1 inhibitors appears to beunmasked in some disease models when combined with Ang2 inhibitors. Dualinhibition of Ang1 and Ang2 cooperatively suppresses ovarian follicularangiogenesis and tumor xenograft growth; however, Ang1 inhibition failsto augment the activity of Ang2 inhibition in suppressing tumorendothelial cell proliferation, corneal angiogenesis, and oxygen-inducedretinal angiogenesis. In no case was Ang1 inhibition shown to 1) confersuperior activity to that of Ang2 inhibition or dual Ang1/Ang2inhibition or 2) antagonize the effects of Ang2 inhibition. Theseresults imply that Ang1 plays a context-dependent role in promotingpostnatal angiogenesis and angiogenesis-associated pathology. Ang1 playsan important role in developmental angiogenesis, but its function inpostnatal neovascularization is less clear. Ang1 has been shown tomediate both pro- and anti-angiogenic effects in various postnatalsettings. To investigate the function of Ang1 by inhibiting endogenousAng1. To that end, we have developed Ang1-neutralizing peptibodies andtested them alone or in combination with Ang2 inhibitors in preclinicalmodels of postnatal angiogenesis.

We generated Ang1-neutralizing peptibodies to investigate the functionalrole of Ang1 in angiogenesis. Phage display peptide libraries werepanned to identify peptides that bound Ang1, but not Ang2. The resultingclones were converted into peptibodies by expressing the peptides in E.coli as fusions to the Fc portion of human IgG1. Peptibodies were thenscreened by enzyme-linked immunosorbent assay (ELISA) and homogeneoustime-resolved fluorescence (HTRF) assays for their ability to neutralizethe interaction between Tie2 and angiopoietins. One of these peptibodieswas affinity-matured to increase its ability to antagonize Ang1, and aresultant peptibody, mL4-3, was chosen for the studies herein. mL4-3exhibited similar potency against several Ang1 orthologs, and itdisplayed>40,000-fold selectivity over Ang2 (Tables 10 and 11). Alsoshown in Table 10 are two previously described peptibodies: AMG 386[also known as 2xCon4 (C)] and L1-7(N). L1-7(N) is a very potent andselective Ang2 inhibitor, and AMG 386 is a dual inhibitor of Ang1 andAng2. The pharmacokinetic profiles of mL4-3 in rodents were acceptablefor daily to weekly s.c. dosing (Table 3).

mL4-3 can be used as a reagent for interrogating Ang1 function in vivo.To assess whether mL4-3 was capable of selectively sequestering Ang1 invivo, mL4-3, L1-7(N), and Fc were administered s.c. to mice, followed byan i.v. challenge with recombinant Ang1. Ang1 induced Tie2phosphorylation in mouse lung endothelium (approximately 5-fold), aneffect that could be prevented by mL4-3, but not by L1-7(N) or Fc (FIG.7).

Next, we wanted to determine whether mL4-3 could neutralize endogenousAng1 in a setting in which Ang1 was known to play a physiologicallyrelevant role. Developmental genetic knockout studies have shown thatAng1 deletion reduces cardiac size and endocardial folding in embryos.In an attempt to replicate this phenotype pharmacologically, mL4-3 wasadministered to pregnant mice in early and middle gestation. Embryoswere harvested at embryonic day 12.5, the time at which lethality wasobserved in Ang1-null mouse embryos. Pharmacokinetic assessment of mouseembryo lysates demonstrated a mean mL4-3 trough level of 3.0 μg/g oftissue, confirming that mL4-3 was capable of crossing the placenta.Histological analysis revealed reduced cardiac size and trabeculation,similar to, but less dramatic than that observed in Ang1-null embryos(FIG. 8). The less pronounced phenotype of the mL4-3 treated embryos maybe a consequence of suboptimal embryonic mL4-3 exposures and incompleteAng1 sequestration. Nonetheless, mL4-3 clearly induces embryonic cardiacdefects that phenocopy those of Ang1 genetic knockout mice, confirmingthe utility of mL4-3 as a reagent for investigating Ang1 function invivo.

Ang1 antagonism augments Ang2 antagonism in suppressing tumor growth. Ina previous report, we demonstrated that systemically administeredL1-7(N) and AMG 386 were capable of inhibiting the growth of Colo205tumor xenografts implanted into nude mice. In that study, the antitumoreffects of AMG 386 were modestly superior to those of L1-7(N) (P=0.006).To confirm that dual Ang1/Ang2 inhibition confers better tumor growthsuppression than Ang2 inhibition alone, a similar experiment wasperformed, but this time groups treated with mL4-3 or a combination ofmL4-3 and L1-7(N) were also tested (FIG. 9). The AMG 386 treatment groupand the mL4-3/L1-7(N) combination treatment group showed comparableantitumor efficacy; moreover, both groups exhibited efficacy superior tothat mediated by either L1-7(N) or mL4-3 alone. In fact, mL4-3 had nodiscernable single-agent effect on tumor growth, implying that combiningAng2 antagonism with Ang1 antagonism may have unmasked the antitumoreffect of Ang1 inhibition. Additional replicates of these experimentsconfirmed that AMG 386 and the mL4-3/L1-7(N) combination mediatedgreater tumor growth suppression than L1-7(N) alone (data not shown).However, in a minority of instances, these differences did not reachstatistical significance, perhaps reflecting the subtle nature of theincremental advantage conferred by dual Ang1/Ang2 inhibition overselective Ang2 inhibition. Selective Ang1 inhibition had no antitumoreffect on its own in any of the experiments in which it was tested (FIG.9 and data not shown).

Ang2 antagonism, but not Ang1 antagonism, inhibits tumor endothelialcell proliferation, corneal angiogenesis, and retinal angiogenesis. Wepreviously showed that dual Ang1/Ang2 inhibition was capable ofsuppressing Colo205 tumor endothelial cell proliferation in vivo. Toinvestigate whether this effect was conferred through Ang1 inhibition,Ang2 inhibition, or a combination of the two, Colo205 tumor-bearing micewere treated with mL4-3, L1-7(N), mL4-3/L1-7(N), or AMG 386. As with thetumor volume readout described in the previous section, mL4-3 had nosingle-agent effect on tumor endothelial cell proliferation, whileL1-7(N) was inhibitory (FIG. 10A). Curiously, however, dual Ang1/Ang2inhibition conferred no greater effect on endothelial cell proliferationthan Ang2 inhibition alone (FIG. 10A), an observation that has beenrepeatedly reproduced (data not shown) and stands in contrast to theapparently cooperative effects of combined Ang1/Ang2 inhibition onColo205 tumor growth. This dissimilarity implies that repression ofendothelial cell proliferation is only one component underlying thetumor growth inhibition mediated by angiopoietin antagonism.

These agents were next tested in two models of ocular angiogenesis, oneinvolving the cornea and the other involving the retina. The cornea isnormally avascular, but pathological angiogenesis can occur in thecornea secondary to conditions such as keratitis and corneal transplantrejection. VEGF- and basic fibroblast growth factor (bFGF)-inducedmodels of corneal angiogenesis were used to test the roles of Ang1 andAng2 antagonism in neovessel formation. As observed with endothelialcell proliferation, corneal angiogenesis appeared to be dependent onAng2, but not on Ang1 (FIGS. 10B and 10C). The same conclusion could bedrawn from evaluation of these angiopoietin-antagonizing peptibodies ina Tie2-dependent retinal model of angiogenesis in whichneovascularization was induced by changes in ambient oxygen tension(FIG. 10D). Thus, in three preclinical settings (endothelial cellproliferation, corneal angiogenesis, and retinal angiogenesis), Ang2inhibition dramatically suppressed neovessel formation, while Ang1inhibition had no effect alone or in combination with Ang2 inhibition.

Selective inhibition of Ang1 or Ang2 induces ovarian atrophy, but notepiphyseal plate thickening. To assess the effects of angiopoietininhibition in normal animals, rats were treated systemically with mL4-3,L1-7(N), or AMG 386 for one month. AMG 386, like VEGF antagonists, hasbeen observed to induce epiphyseal plate thickening and ovarian atrophy,effects considered to be mechanism-based consequences of antiangiogenictherapy. In the present study, AMG 386 provoked epiphyseal platethickening in all treated animals, while, remarkably, L1-7(N) and mL4-3failed to alter epiphyseal morphology in any rats (Table 13). Thus,induction of epiphyseal plate thickening appears to require inhibitionof both Ang1 and Ang2. In striking contrast, all three peptibodiesproduced ovarian atrophy at similar incidence rates, indicating thatselective inhibition of Ang1 or Ang2 is sufficient to induce ovarianatrophy.

Ang1 and Ang2 Inhibitors Cooperatively Suppress Ovarian FollicularAngiogenesis. To better understand the effects of angiopoietininhibition on the ovary, we employed a hormone-induced model of ovarianfollicular angiogenesis that allowed controlled assessment ofneovascularization in mice that had never previously ovulated. In thismodel, pregnant mare serum (PMS) and human chorionic gonadotropin (HCG)were used to induce rapid, synchronized ovulation in multiple follicles(FIG. 11). Mice were treated systemically with Fc control, mL4-3,L1-7(N), or an mL4-3/L1-7(N) combination to determine the effects ofthese agents on neovessel formation in transforming Graafian follicles.Two identically-designed replicates of this experiment were performed ondifferent days, and remarkably, both yielded almost identical activityprofiles with respect to percentage inhibition of blood vessel area(replicate 1, replicate 2): L1-7(N) (8%, 11%), mL4-3 (15%, 14%),mL4-3/L1-7(N) (24%, 26%). All single-agent and combination peptibodygroups, with the exception of the L1-7(N) group in Experiment 1,mediated statistically significant inhibition of angiogenesis relativeto the Fc control (P<0.05) (FIG. 11). Thus, both inhibition of ovarianangiogenesis and induction of ovarian atrophy could be elicited byinhibiting Ang1, Ang2, or both, consistent with the notion that theobserved ovarian atrophy was a consequence of failed neovesseldevelopment.

We demonstrate that Ang1 inhibition plays a context-dependent role inthe suppression of angiogenesis in preclinical disease models and innormal animals. In utero, pharmacologic Ang1 inhibition partiallyphenocopied the genetic ablation of Ang1, consistent with the importantrole of Ang1 in developmental angiogenesis. Postnatally, selective Ang1antagonism inhibited ovarian angiogenesis and induced ovarian atrophy,effects that could also be achieved by inhibiting Ang2 alone or Ang1plus Ang2 together. However, in postnatal disease models, Ang1inhibition had little effect on its own, although its biologicalactivity appeared to be unmasked in some settings when combined withAng2 suppression. The mechanism underlying the differential dependencyon Ang1 in these settings remains to be determined

The ovary, by virtue of its role in reproductive cycling, is one of thefew organs that undergoes normal angiogenesis in adults. Based on theovarian expression patterns of Ang1 and Ang2 in hormone-inducedovulating rats, it has been proposed that Ang2 plays an early role invessel invasion, and Ang1 plays a later role to mature the newly-formedvessels. Under this hypothesis, Ang2 and Ang1 perform opposingfunctions, where Ang2 initially displaces Ang1 from Tie2, resulting invessel destabilization and angiogenesis. This state of plasticity issubsequently reversed when Ang1 ousts Ang2 from the receptor tore-establish vascular quiescence and stability. In conflict with thismodel, the data from the current study imply that Ang1 and Ang2 bothplay pro-angiogenic roles in the ovary.

In the Colo205 tumor xenograft model, antagonism of Ang1 and Ang2mediated greater tumor suppression than was achieved by inhibiting Ang1or Ang2 individually, indicating that this model is dependent on bothangiopoietins. However, in the same model, only Ang2 inhibition wascapable of down-modulating tumor endothelial cell proliferation,suggesting that Ang1 is not involved in this function. What accounts forthe different dependencies of these two endpoints on Ang1? Onepossibility is that Ang1 inhibition has a direct effect on tumor cells.This seems unlikely, however, given that AMG 386, a dual inhibitor ofAng1 and Ang2, has no effect on the in vitro growth of cultured Colo205tumor cells. A second possibility is that Ang1 antagonism plays ananti-angiogenic role that is not conferred through inhibition ofendothelial cell proliferation, but instead through mechanisms thatmight impact functions such as endothelial cell migration or invasion.This explanation could be applicable if Ang1 and Ang2 mediatedqualitatively or quantitatively different signals through Tie2, or ifAng1 signaled through additional receptors that were not responsive toAng2. A third possibility is that Ang1 signals through Tie2 onnon-endothelial cells, such as Tie2-expressing monocytes (TEMs). TEMsare recruited to tumors, where they cluster around neovessels. Selectiveablation of TEMs in tumor-bearing mice suppresses tumor angiogenesis andinhibits tumor growth, and it has been postulated that TEMs promotetumor angiogenesis by providing paracrine signals that stimulateneovessels. Perhaps Ang1 stimulates TEMs to release pro-angiogeniccytokines other than the angiopoietins. In such a setting, inhibition ofAng1 could have an indirect anti-neovascular effect that mightcomplement the direct anti-angiogenic effect of Ang2 suppression.

In contrast to the subtle and context-dependent effects of Ang1inhibition, Ang2 inhibition frequently mediated effects that wereequivalent or nearly equivalent to those conferred by combinedantagonism of Ang1 and Ang2, implying that Ang2 may be the dominantangiopoietin involved in postnatal angiogenesis. Ang1 appears to be thedominant angiopoietin involved in prenatal angiogenesis, suggesting ashift in the dependency on these two factors around the time of birth.Our inhibitors do not antagonize Ang4, but the functional relevance ofthis factor is unclear, given its lung-restricted expression pattern.

Ang1 and Ang2 have been shown to play both similar and opposingfunctional roles in various in vitro and in vivo systems. The inabilityto draw consistent conclusions in this regard across multiplepublications may be in part a consequence of the different conditionsunder which the question was examined. These differences includeevaluation of 1) in vitro versus in vivo systems, 2) prenatal versuspostnatal angiogenesis, 3) varying vascular beds, 4) pathological versusnormal angiogenesis, and 5) gain-of-function versus loss-of-functionexperimental designs. This final difference may be particularlyimportant, as the addition of exogenous factors to a model system may bea less physiologically relevant means to elucidate function than removalof endogenous factors. Perhaps the most informative publishedexperiments in this regard are those in which Ang1 and Ang2 have beengenetically deleted in the germline of rodents. These studies providesignificant insight into the developmental roles of Ang1 and Ang2.However, it is more difficult to genetically examine the postnatal invivo function of Ang1 and Ang2 without the availability of conditionalknockout systems; the constitutive Ang1 knockout mouse dies in utero (asdoes the constitutive Ang2 knockout on some strain backgrounds), and thepostnatal phenotype of surviving Ang2 knockout mice may be influenced byresidual effects of developmental gene deletion. By using pharmacologicAng1 and Ang2 inhibitors to examine the postnatal roles of Ang1 and Ang2in vivo, we have circumvented these issues. The results of the currentstudy imply that Ang1 and Ang2 do not functionally oppose one another inpostnatal systems, and in some cases, they appear to act cooperatively.

Pathological angiogenesis is associated with altered angiopoietin levelsin a number of diseases, including cancer, diabetic retinopathy, maculardegeneration, rheumatoid arthritis, osteoarthritis, and psoriasis.Angiopoietin-targeted interventions in these therapeutic indications mayprovide clinical benefit. The data presented herein suggest that, insome settings, combined inhibition of Ang1 and Ang2 may provide superiortherapeutic efficacy to that mediated by targeting Ang2 alone.

Methods Phage Display Selection of Ang1-Binding Peptides.

Three filamentous phage libraries, TN8-IX (5×10⁹ independenttransformants), TN12-I (1.4×10⁹ independent transformants), and Linear(2.3×10⁹ independent transformants) (Dyax Corp., Cambridge, Mass.), wereused to select for Ang1-binding phage. After negative selection on emptystreptavidin Dynabeads (Invitrogen Corporation, Carlsbad, Calif.)blocked with 2% bovine serum albumin (BSA) or beads loaded withbiotinylated Ang2 (R&D Systems, Inc., Minneapolis, Minn.), remainingphage were incubated with beads loaded with biotinylated Ang1 (R&DSystems, Inc.). After extensive washing, the phage from each round ofselection were eluted in a nonspecific manner using 100 mM triethylaminesolution (Sigma-Aldrich Inc., St. Louis, Mo.). The eluted phage wereamplified in E. coli strain XL-1 Blue MRF′, purified by precipitation,and then used for the next round of selection.

After three rounds of selection, individual phage clones were isolatedand analyzed by phage ELISA and DNA sequencing. Briefly, Ang1 proteinwas coated on 96-well Maxisorp plates (Nunc brand, Thermo FisherScientific, Rochester, N.Y.) and blocked with PBST (PBS with 0.05%Tween-20) containing 4% dry milk. Phage supernatants were incubated inthe wells and bound phage were detected with an HRP-conjugated anti-M13antibody (Amersham Pharmacia Biotech, Piscataway, N.J.). To checkcross-reactivity to Ang2 or streptavidin, control plates were set up ina similar fashion. ELISA results and DNA sequencing data were used ascriteria for selecting peptide sequences to express in a peptibodyformat. Peptibodies were evaluated in an HTRF assay, and several werechosen for affinity maturation.

Peptide affinity maturation was performed by generating and panningnucleotide-doped phage display libraries. Libraries with over 1×10⁹independent transformants were obtained. These focused libraries werepanned by a procedure similar to that used for panning the primarylibraries.

Peptibody Expression and Purification.

Peptibody mL4-3 was expressed and purified as described in Oliner, J.,et al. 2004., Cancer Cell 6:507-516. The amino acid sequence of mL4-3 isas follows, where Fc in bold italics denotes the human IgG1 Fc sequenceas described previously in Oliner, J., et al. 2004., Cancer Cell6:507-516:

(SEQ ID NO: 47) MREWTEEMQVIFDAMMFGPRNDRGGSGSATGSGSTASSGSGSATHREWTEEMQVIFDAMMFGPRNDRGGGGG-

The amino acid sequence of the Fc portion of the peptibody mL4-3 is asfollows (from amino terminus to carboxyl terminus):

(SEQ ID NO: 48) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

Angiopoietin: Tie2 Neutralization HTRF Assay.

Europium-labeled streptavidin (LANCE reagent, PerkinElmer Inc., Boston,Mass.) and biotinylated human Ang1 (R&D Systems, Inc.) or Ang2 weremixed in HTRF buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.05% Tween20, 0.1% BSA) and incubated at room temperature in the dark for 30minutes on a shaker. Equal volumes of the above mixture and seriallydiluted peptibodies or Fc were mixed and incubated for 1 hour at roomtemperature. Equal volumes of allophycocyanin-conjugated Tie2-Fc(Tie2-APC) (Prozyme, San Leandro, Calif.) and the above mixture weremixed and incubated for 2 hours at room temperature. The finalconcentrations of reagents in the assay were 4 nM europium-streptavidin,2 nM biotinylated Ang1 or Ang2, and 5 nM Tie2-APC. Peptibodies wereserially diluted from 10,000 nM to 0.5 nM or 100 nM to 0.005 nM togenerate full titration curves. Neutralization of angiopoietin:Tie2interaction was measured by the diminishing energy transfer between APCand europium and was quantified using a Rubystar plate reader (BMGLabtechnologies, Offenberg, Germany). The potency of angipoietin/Tie2neutralization was determined by calculating the percentage inhibitionof each peptibody dilution in reference to the maximum (no angiopoietinin the assay mixture) and minimum inhibition (no peptibody in the assaymixture) controls. IC₅₀ values were calculated by plotting percentageinhibition using XLfit4, where fit=A+((B−A)/(1+((C/X)̂D))) (IDBS,Guildford, UK).

Angiopoietin:Tie2 Neutralization ELISA.

Ninety-six-well microtiter plates were coated with a panel ofrecombinant angiopoietins in 293T cell conditioned media (DMEM/50 ug/mlBSA) at 37° C. for 1 hour. The conditioned media were used atangiopoietin concentrations that conferred 70% of maximally achievablebinding to 1 nM hTie2-Fc (Recombinant hTie2-Fc, Catalog #313-TI, R&DSystems Inc.). Plates were washed three times with PBS/0.1% Tween-20 andthen block for 2 hours at room temperature with PBS/5% BSA. The blockingsolution was removed without washing the plates. mL4-3 or Fc seriallydiluted in a solution of 1 nM Tie2-Fc/1% BSA/PBS was added to theangiopoietin-coated plates, which were incubated overnight at roomtemperature and then washed with PBS/1% Tween-20. A mouse-derivedanti-Tie2 antibody (Catalog #557039, BD Pharmingen Inc., San Jose,Calif.) was added to each well at a final concentration of 1 ug/ml andincubated for 1 hour at room temperature. Plates were then washed 3times with PBS/0.1% Tween-20. Goat anti-mouse-IgG-HRP (Horseradishperoxidase-conjugated goat anti-mouse antibody, Catalog #31432, Pierce,Rockford, Ill.) was added at a dilution of 1:10,000 in PBS/1% BSA toeach well and the plates were incubated for 1 hour at room temperature.Plates were washed three times with PBS/0.1% Tween-20 before TMBsubstrate (SureBlue Reserve TMB, Catalog#53-00002, KPL, Gaithersburg,Md.) was added and optical density at 650 nM was measured on a platereader (SpectraMax, Molecular Devices, Sunnyvale, Calif.). The degree ofangiopoietin:Tie2 neutralization (IC₅₀) was determined by comparisonagainst a Tie2 standard curve (the binding activity of serially dilutedTie2 in the absence of competitor) using XLfit.

Animal Studies.

All procedures were approved by the Amgen Animal Care and Use Committeeand met Association for Assessment and Accreditation of LaboratoryAnimal Care standards.

Pharmacokinetic Assessment.

Three CD-1 mice received a single s.c. injection of 3.2 mg/kg of mL4-3,and two Sprague-Dawley rats received a single i.v. injection of 10 mg/kgof mL4-3. Blood samples were collected up to 274 hours from the mice and336 hours from the rats for serum pharmacokinetic assessment. mL4-3concentrations in serum samples from each species were measured by anenzyme-linked immunosorbent assay (ELISA). Polystyrene 96-well plateswere coated with human Ang1, followed by incubation withmL4-3-containing serum samples. After washing away any unboundsubstances, a horseradish peroxidase-labeled monoclonal mouse anti-IgG1antibody was added to the wells. Following a wash step to remove anyunbound monoclonal antibody, TMB-peroxidase substrate was added to thewells. The optical density units measured at 450-650 nm were convertedto concentrations by comparing to a concurrently analyzed standardcurve.

Pharmacokinetic parameters were calculated by noncompartmental analysisof the individual serum concentration-time data (WinNonlin Professional,version 3.3; Pharsight Corp, Mountain View, Calif.). Terminal phasehalf-life (t_(1/2)) was calculated as t_(1/2)=ln(2)/λ_(z), in whichλ_(z) is the first-order terminal phase elimination rate constantestimated via linear regression of the terminal log-linear decay phase.Area under the serum concentration-time curve (AUC_(0-last)) wasestimated by the linear/log trapezoidal method from time 0 to the timeof the last quantifiable concentration (C_(last)). AUC_(0-inf) wasestimated from time 0 to infinity asAUC_(0-inf)=AUC_(0-last)+C_(last)/λ_(z). AUC_(0-inf) values werenormalized to a 1 mg/kg dose.

Because the pharmacokinetic properties of mL4-3, L1-7, and AMG 386 weredissimilar, the dose levels and schedules of each agent were chosen,where possible, to achieve equimolar serum steady-state C_(min)concentrations within pharmacology studies.

Administration of mL4-3 to Pregnant Mice.Two groups of six 129/SV female mice were impregnated by C57BL/6 males.Pregnant females were dosed with 300 mg/kg Fc control or mL4-3 by s.c.administration on gestational days E4.5, E7.5 and E11.5. Conceptuses(embryos and placentae) were removed on day E12.5, evaluated for grossabnormalities, and fixed by immersion in IHC-zinc (mL4-3-treated, n=10;Fc control-treated, n=10) or Bouin's solution (mL4-3-treated, n=5; Fccontrol-treated, n=6). Paraffin-embedded tissues were step-sectioned at50-1.1m intervals through the heart (embryos in both longitudinal andtransverse orientation) and the middle of the placenta. Serial sectionsfrom each interval were stained with hematoxylin and eosin (H&E) orstained with a conventional indirect immunohistochemistry procedureusing polyclonal anti-CD31 (rat anti-mouse monoclonal MEC 13.3, BDBiosciences Pharmingen, San Diego, Calif.) to specifically label bloodvessels. Criteria for scoring changes were established by evaluatingsections with a foreknowledge of the treatment. Subsequently, lesionseverity was graded rapidly using a tiered scale (minimal, mild,moderate, or marked) and a blinded analytical paradigm. These ordinalpathology data were analyzed using the Chi-square test contained in theJMP statistical software package (v.5.1; SAS Institute Inc., Cary,N.C.). An embryo from each pregnant mother collected on day E12.5 wasanalyzed by ELISA using human Ang1 as a capture reagent and horseradishperoxidase-labeled monoclonal mouse anti-IgG1 antibody as a detectionreagent.

Tie2 Phosphorylation Assay.

The effect of the selective angiopoietin inhibitors on Ang1-induced Tie2phosphorylation in mouse lungs was performed as described in Hodous, B.L., et al. 2007., J. Med. Chem. 50:611-626). Briefly, CD-1 nude mice(Charles River Laboratories, Wilmington, Mass.) were treated s.c. oncedaily for 23 days with Fc control (20 mg/kg), mL4-3 (20 mg/kg) orL1-7(N) (2 mg/kg). Mice (n=3 per group) were then administered 12 μg byi.v. injection of recombinant Ang1 (R&D Systems Inc.). Fifteen minuteslater, mouse lungs were harvested, and the levels of phosphorylated Tie2were determined by immunoprecipitation-Western blot analysis.Statistical analysis was performed using analysis of variance (ANOVA)followed by Fisher's post hoc test using StatView 5.0.1 software (SASInstitute Inc.). Results are expressed as mean±standard error (SE).

Tumor Xenograft Models.

Eight- to 10-week old female CD1 nude mice (Charles River Laboratories)were used in all experiments. Mice were injected s.c. with 2×10⁶ Colo205cells in one-third volume Matrigel (BD Biosciences, San Jose, Calif.).Peptibodies or Fc control were administered by s.c. injection oncetumors were established. AMG 386 was dosed twice per week; the otherpeptibodies and Fc control were dosed once daily. Where necessary, Fccontrol protein was added to the treatment groups to match the totalamount of protein delivered in the combination group (5.2 mg/kg). Tumormeasurements and body weights were recorded twice per week. All tumorstudies were performed in a blinded fashion. Tumor volume was calculatedas length×width×height in mm³ Results are expressed as mean±SE.Statistical analysis was performed using repeated measures analysis ofvariance followed by a Scheffe post hoc test using StatView 5.0.1software (SAS Institute Inc.).

Tumor Endothelial Cell Proliferation Assay.

Tumor endothelial cell proliferation was assayed as previously described(Oliner, J., et al. 2004., Cancer Cell 6:507-516). Briefly, Colo205tumor-bearing mice were treated systemically with peptibodies for 72hours and implanted with osmotic pumps containing 3 mg/mL BrdU 16 hoursprior to euthanasia. Tumors were harvested, dissociated, fixed, andstained to allow determination of BrdU incorporation in tumorendothelial cells. Statistical analysis was performed using an unpairedt-test.

Corneal Angiogenesis Model.

VEGF and bFGF-induced angiogenesis studies were performed in female CDrats (n=8 per group) as described in Coxon, A., et al. Arthritis Rheum46:2604-2612, 2002. Inhibition of interleukin-1 but not tumor necrosisfactor suppresses neovascularization in rat models of cornealangiogenesis and adjuvant arthritis. Treatment with Fc (60 mg/kg),L1-7(N) (5 mg/kg), mL4-3 (60 mg/kg), or the combination of L1-7(N) andmL4-3 (at the same doses used in the single-agent groups) was initiatedon the day prior to surgery and continued on day 3 and day 6. On day 8the study was terminated and the corneas were photographed, as described(Oliner, J., et al. 2004., Cancer Cell 6:507-516). For each cornealimage, the number of blood vessels intersecting the midpoint between theimplanted disc and the limbus was counted. All evaluations wereperformed in a blinded fashion. Statistical significance was assessed byANOVA followed by Fisher's post hoc test.

Retinal Neovascularization.

Ischemic retinopathy was produced in C57BL/6J mice using the methoddescribed by Smith et al., Invest. Ophthalmol V is Sci 35: 101-111,1994. Postnatal day seven (P7) pups and their mothers were placed in ahyperoxic chamber (75±0.5% oxygen) for 5 days and then returned to roomair for an additional 5 days (n=7 pups per group). Chamber temperaturewas maintained between 20° C. and 22° C., and oxygen was constantlycontrolled by an oxygen control unit (ProOx Model P110 coupled to anoxygen sensor Model E702 Biospherix Ltd, Redfield, N.Y.). One cage withP7 pups remained at room air (normoxia condition). Fc control (200mg/kg), mL4-3 (100 mg/kg), L1-7(N) (100 mg/kg), or mL4-3/L1-7(N)combination (100 mg/kg each) was administered s.c. once daily for ninedays starting on P8. From P8 to P11 the injections were administeredusing ports to gain access into the chamber. On P17 the pups weresacrificed and their eyes removed and fixed using Davidson's fixative.The eyes were then processed into paraffin using standard methods. Stepsections were cut parallel to the optical axis at 100-μm intervals. Theblocks were completely through-sectioned, resulting in 15 or 16 sectionsper eye. All sections were stained with H&E. Of the 15 or 16 slides inthe step-section series, the middle 10 consecutive slides were used inthe analyses, bracketing either side of the optical axis. For eachsection, the number of vascular nuclei (both endothelial and pericytenuclei) that were on the vitreous side of the inner limiting membranewere counted. The individual slide counts were recorded and all tensection counts summed for each animal. Five mice in each study groupwere counted. All counts were performed in a blinded fashion, withoutknowledge of treatment conditions. Statistical analysis was performed byANOVA followed by Fisher's post hoc test.

Ovarian Follicular Angiogenesis.

Superovulation was induced in study mice using standard methodology.Briefly, four-week old female C57BL/6J mice were injected with 5-7 IUPMS, effectively resetting the estrus cycle. Forty-eight hours later,the mice were injected with 5 IU of HCG to induce superovulation. Thefemales were then faux-bred and remained on study for 24 hours. Studymice were treated with peptibodies twice per day. Dosing commenced atthe time of the initial PMS injection and continued for two consecutivedays, with the fourth dose given concurrently with the HCG injection.Mice were euthanized 48 hours following the HCG injection. Right andleft ovaries were removed and immersion-fixed in cold zinc trissolution. After 48 hours, ovaries were transferred to 70% ethanol andprocessed to paraffin using standard methods. Two sequential sectionswere cut from each ovary pair and individually stained either with H&Eor immunostained for vascular endothelium (CD31, rat anti-mousemonoclonal MEC 13.3, BD Biosciences Pharmingen) using DAB as thechromogen. Additionally, the anti-CD31 IHC sections were lightlycounterstained with hematoxylin. The individual follicles selected foranalysis were identified based on transformational state. This wasdetermined by treatment-blind inspection of the H&E sections under lowpower. Corresponding images of ten transformed follicles per animal,where feasible, were then captured at 10× objective magnification fromthe anti-CD31 immunostained sections. The follicle section area wasdelineated as a ROI, and the CD31-positive area fraction was determinedvia RGB thresholding using MetaMorph image analysis software (MetaMorphv6.1, UIC, Downingtown, Pa.). Statistical analysis was performed byANOVA followed by Dunnett's post hoc test.

Evaluation of Normal Tissues in Treated Rats.

Peptibodies were evaluated in Sprague-Dawley rats (Charles RiverLaboratories) for effects on normal tissues. Animals received 300 mg/kgof AMG 386, L1-7(N) or mL4-3 IV twice weekly for 28 days (n=10 animalsper group). At scheduled necropsy, a full tissue set was sectioned,stained, and observed for microscopic changes.

TABLE 10 Peptibodies competitively inhibit angiopoietin: Tie2interactions hAng1 hAng2 Agent IC₅₀ (nM) IC₅₀ (nM) L1-7(N) >10,000 0.064mL4-3 0.022 3085 AMG 386 6.2 0.029 Fc >10,000 >10,000 h, human

TABLE 11 mL4-3 selectively neutralizes Ang1:Tie2 interactions hAng1mAng1 rAng1 cAng1 hAng2 mAng2 cAng2 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀Agent (nM) (nM) (nM) (nM) (nM) (nM) (nM) mL4-3 0.045 0.033 0.061 0.0391876 >10,000 1890Fc >10,000 >10,000 >10,000 >10,000 >10,000 >10,000 >10,000 h, human; m,mouse; r, rabbit; c, cynomolgus monkey

TABLE 12 Mean pharmacokinetic parameters of angiopoietin inhibitors inmice and rats Mouse Rat Dose-normalized Dose-normalized t_(1/2)AUC_(0-inf) t_(1/2) AUC_(0-inf) Agent (hr) (μM · hr/mg/kg) (hr) (μM ·hr/mg/kg) mL4-3 45 5.0 42 3.6 L1-7(N)^(a) 56 7.0 47 4.6 AMG 386^(a) 9715 85 8.7 ^(a)Adapted from Oliner et al (18).

TABLE 13 Selective inhibition of Ang1 or Ang2 induces ovarian atrophy,but not epiphyseal plate thickening Epiphyseal plate Epiphyseal plateAgent thickening (males) thickening (females) Ovarian atrophy AMG 386 1010 8 L1-7(N) 0 0 8 mL4-3 0 0 6 n = 10 per group

1. An isolated antibody which comprises a heavy chain variable domain having the sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; wherein said antibody specifically binds to at least one of Ang1 and Ang2 ligands of Tie 2 receptor.
 2. An isolated antibody which comprises a light chain variable domain having the sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; wherein said antibody specifically binds to at least one of Ang1 and Ang2 ligands of Tie 2 receptor.
 3. An isolated antibody which comprises a heavy chain variable domain and a light chain variable domain, wherein said heavy chain is comprised of 3 CDRs and said light chain is comprised of 3 CDRs, wherein the sequences of said CDRs of said antibody are selected from the group consisting of: (a) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (b) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (c) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20, 27, 36 of the LC, (d) SEQ ID NOs: 18, 26, 37 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (e) SEQ ID NOs: 18, 26, 38 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (f) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (g) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 21, 27, 33 of the LC, (h) SEQ ID NOs: 18, 28, 39 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (i) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 22, 27, 33 of the LC, (j) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 22, 27, 33 of the LC, (k) SEQ ID NOs: 18, 29, 39 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (l) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 23, 27, 33 of the LC, (m) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20, 27, 40 of the LC, (n) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 21, 27, 33 of the LC, (o) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 24, 27, 33 of the LC, (p) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 21, 27, 33 of the LC, (q) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 23, 27, 33 of the LC, (r) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20, 30, 33 of the LC, (s) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 25, 27, 33 of the LC, (t) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20, 30, 33 of the LC, (u) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20, 27, 40 of the LC, and (v) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20, 31, 33 of the LC; wherein said antibody specifically binds to at least one of Ang1 and Ang2 ligands of Tie 2 receptor.
 4. The isolated antibody of claim 3, wherein said antibody is selected from the group consisting of: H6L7, H5L7, H4L13, H11L7, H10L7, H4L7, H5L6, H2L7, H5L8, H6L8, H3L7, H5L4, H4L12, H6L6, H4L2, H4L6, H4L4, H5L11, H5L1, H4L11, H5L12, and H5L9.
 5. The isolated antibody of claim 4 that is a fully human antibody.
 6. An antibody fragment of the antibody of claim 3 which comprises a CDR region with the amino acid sequence selected from a group consisting of: SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:
 40. 7. An isolated nucleic acid molecule encoding the antibody or the antibody fragment of claim 1 or
 6. 8. A vector containing a nucleic acid molecule of claim
 7. 9. A host cell containing the vector of claim
 8. 10. The host cell of claim 9 that is a CHO cell.
 11. A method of making the antibody of claim 4 which comprises expressing said antibody in a host cell.
 12. The method of claim 11 wherein said host cell is a CHO cell.
 13. A pharmaceutical composition comprising any one of the antibodies selected from the group consisting of H6L7, H5L7, H4L13, H11L7, H10L7, H4L7, H5L6, H2L7, H5L8, H6L8, H3L7, H5L4, H4L12, H6L6, H4L2, H4L6, H4L4, H5L11, H5L1, H4L11, H5L12, and H5L9; in admixture with a pharmaceutically acceptable carrier therefor.
 14. The pharmaceutical composition of claim 13 further comprising a molecule selected from the group consisting of a reporter molecule, a water soluble polymer, an antibody Fc region, and a cytotoxic agent.
 15. The pharmaceutical composition of claim 14, wherein said pharmaceutically acceptable carrier is a pharmaceutical formulation agent.
 16. A method of inhibiting undesired angiogenesis that comprises administering to a subject in need thereof, an effective amount of any one of the antibodies selected from the group consisting of H6L7, H5L7, H4L13, H11L7, H10L7, H4L7, H5L6, H2L7, H5L8, H6L8, H3L7, H5L4, H4L12, H6L6, H4L2, H4L6, H4L4, H5L11, H5L1, H4L11, H5L12, and H5L9.
 17. The method of claim 16, wherein said undesired angiogenesis is cancer. 